Inhibitors of lysine gingipain

ABSTRACT

The present invention relates generally to therapeutics targeting the bacterium  Porphyromonas gingivalis , including its protease Lysine gingipain (Kgp), and their use for the treatment of disorders associated with  P. gingivalis  infection, including brain disorders such as Alzheimer&#39;s disease. In certain embodiments, the invention provides compounds according to Formula I, as described herein, and pharmaceutically acceptable salts thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/683,348 filed on Aug. 22, 2017; which is a division of U.S.patent application Ser. No. 14/875,416 filed on Oct. 5, 2015, now U.S.Pat. No. 9,758,473 issued on Sep. 12, 2017; which claims priority toU.S. Provisional Pat. Appl. No. 62/060,483 filed on Oct. 6, 2014, whichapplications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Infection with the bacteria Porphyromonas gingivalis has been linked tothe development of periodontal disease, Alzheimer's and other braindisorders, cardiovascular disease, diabetes, cancer, liver disease,kidney disease, preterm birth, arthritis, pneumonia and other disorders.P. gingivalis is an anaerobic asaccharolytic gram-negative rod bacteriumthat is known to infect the oral cavity and translocate systemicallyinto coronary arteries, aorta, placental tissue, the brain, the kidneys,and the liver. The bacterium has also been identified in canceroustissues and a mechanism has been proposed by which gingipains cantrigger immortalization and metastasis. See: Gandhimadhi, et al. Journalof Indian Society of Periodontology. 2010; 14(2):114-120; Liao, et al.,Med Hypotheses, 2009. 72(6): 732-5; Byrne, et al., Oral MicrobiolImmunol, 2009. 24(6): 469-77; Mahendra, et al., J Maxillofac Oral Surg,2009. 8(2): 108-13; Stelzel, et al., J Periodontol, 2002. 73(8): 868-70;Katz, et al., Journal of Dental Research, 2009. 88(6): 575-578; Poole,et al., J Alzheimers Dis, 2015, 43(1): 67-80; Ishikawa, et al., BiochimBiophys Acta, 2013. 1832(12): 2035-2043; Inaba, et al., CellularMicrobiology, 2014. 16(1): 131-145.

P. gingivalis produces extracellular proteases called gingipains,including Arginine Gingipain A (RgpA), Arginine Gingipain B (RgpB) andLysine Gingipain (Kgp). Gingipains contribute to many functions of theorganism including its survival and virulence. Gingipains can besecreted, transported to outer membrane surfaces of P. gingivalis, orreleased in outer membrane vesicles by the bacterium. Gingipains degradea broad range of proteins (e.g., immunoglobulins, proteinase inhibitors,actin, and collagen) which can lead to cytoskeleton collapse andapoptosis in many types of cells. Recent research has demonstrated thatsmall, peptide-derived inhibitors of Kgp can prevent gingipain-inducedepithelial cell death. See: Travis, et al., Adv Exp Med Biol, 2000. 477:455-65; Sheets, et al., Infect Immun, 2005. 73(3): 1543-52; Sheets, etal., Infect Immun, 2006. 74(10): 5667-78; Stathopoulou, et al., BMCMicrobiol, 2009. 9: 107. New compounds for the inhibition of gingipainactivity and the treatment of diseases associated with gingipainactivity and P. gingivalis infection are needed. The present inventionaddresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound according to Formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Z is a thiol-reactive group or a masked thiol-reactive group;    -   A is selected from —CH₂— and —O—;    -   B and D are independently selected from hydrogen, halogen, C₁₋₄        haloalkyl, and C₁₋₄ haloalkoxy;    -   R¹ is selected from hydrogen and an amine protecting group;    -   R² is hydrogen; and    -   R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₁₋₈ alkyl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, -L-R⁵, and —OR⁶, wherein    -   L is selected from —O—, —NR—, C₁₋₄ alkylene, and 2- to        4-membered heteroalkylene, wherein R is selected from hydrogen        and C₁₋₈ alkyl,    -   R⁵ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₃₋₈ cycloalkyl, and 5-to-12 membered saturated heterocyclyl,        and    -   —OR⁶ and the carbonyl to which it is bonded form an amine        protecting group,    -   and wherein R³ is optionally substituted with one or more        substituents selected from halo, —CN, —NO₂, —N₃, —OH, R^(a),        R^(b), —OR^(a), —OR^(b), —(CH₂)_(k)C(O)R^(c),        —NR^(d)(CH₂)_(u)C(O)R^(c), —O(CH₂)_(u)C(O)R^(c),        —(CH₂)_(k)CONR^(d)R^(d), —(CH₂)_(k)NR^(d)C(O)R^(c),        —NR^(d)(CH₂)_(u)CONR^(d)R^(d), —NR^(d)(CH₂)_(u)NR^(d)C(O)R^(c),        —O(CH₂)_(u)CONR^(d)R^(d), —O(CH₂)_(u)NR^(d)C(O)R^(c),        —(CH₂)_(k)S(O)₂NR^(d)R^(d), —(CH₂)_(k)NR^(d)S(O)₂R^(c),        —(CH₂)_(k)S(O)₂R^(c), —(CH₂)_(k)S(O)R^(c), —(CH₂)_(k)SR^(d),        —NR^(d)(CH₂)_(u)S(O)₂NR^(d)R^(d),        —NR^(d)(CH₂)_(u)NR^(d)S(O)₂R^(c), —NR^(d)(CH₂)_(u)S(O)₂R^(c),        —NR^(d)(CH₂)_(u)S(O)R^(c), —NR^(d)(CH₂)_(u)SR^(d),        —O(CH₂)_(u)S(O)₂NR^(d)R^(d), —O(CH₂)_(u)NR^(d)S(O)₂R^(c),        —O(CH₂)_(u)S(O)₂R^(c), —O(CH₂)_(u)S(O)R^(c), and        —O(CH₂)_(u)SR^(c), wherein:    -   each R^(a) is independently selected from C₁₋₄ alkyl and C₁₋₄        haloalkyl,    -   each R^(b) is independently selected from C₃₋₆ cycloalkyl, C₃₋₆        halocycloalkyl, C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(c) is independently selected from —OH, C₁₋₈ alkyl, C₁₋₈        haloalkyl, C₃₋₈ cycloalkyl, C₃₋₈ halocycloalkyl, C₆₋₁₀ aryl,        (C₆₋₁₀ aryl)-(C₁₋₈ alkyl), 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(d) is independently selected from hydrogen and C₁₋₈        alkyl,    -   each subscript k is independently selected from 0, 1, 2, 3, 4,        5, and 6, and    -   each subscript u is independently selected from 1, 2, 3, 4, 5,        and 6; and    -   R⁴ is selected from hydrogen, halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy,        C₁₋₄ haloalkyl, and C₁₋₄ haloalkoxy;    -   provided that when Z is benzothiazol-2-yl-carbonyl, A is —CH₂—,        and B, D, and R¹ are hydrogen, then R³ is other than benzyloxy,        substituted benzyloxy, or 1-(3-phenyl-propanoyl)piperidin-3-yl,        and    -   provided that when Z is phenoxymethyl-carbonyl or substituted        phenoxymethyl-carbonyl, A is —CH₂—, and B and D are hydrogen,        then R³ is other than (2-phenyl)ethyl or substituted        (2-phenyl)ethyl.

In some embodiments, the compound has a structure according to FormulaIc:

-   -   wherein R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered        heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, and -L-R⁵,    -   wherein L is C₁₋₄ alkylene.

In some embodiments, the compound has a structure according to FormulaId:

-   -   wherein R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered        heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, and -L-R⁵, wherein L is C₁₋₄ alkylene.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound according to Formula I and a pharmaceuticallyacceptable excipient.

In another aspect, the invention provides a method of treating a diseaseor condition associated with P. gingivalis infection. The methodincludes administering to a subject an effective amount of a compoundaccording to Formula Ie:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Z is a thiol-reactive group or a masked thiol-reactive group;    -   A is selected from —CH₂— and —O—;    -   B and D are independently selected from hydrogen, halogen, C₁₋₄        haloalkyl, and C₁₋₄ haloalkoxy;    -   R¹ is selected from hydrogen and an amine protecting group;    -   R² is hydrogen; and    -   R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₁₋₈ alkyl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, -L-R⁵, and —OR⁶, wherein    -   L is selected from —O—, —NR—, C₁₋₄ alkylene, and 2- to        4-membered heteroalkylene, wherein R is selected from hydrogen        and C₁₋₈ alkyl,    -   R⁵ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₃₋₈ cycloalkyl, and 5-to-12 membered saturated heterocyclyl,        and    -   —OR⁶ and the carbonyl to which it is bonded form an amine        protecting group,    -   and wherein R³ is optionally substituted with one or more        substituents selected from halo, —CN, —NO₂, —N₃, —OH, R^(a),        R^(b), —OR^(a), —OR^(b), —(CH₂)_(k)C(O)R^(c),        —NR^(d)(CH₂)_(u)C(O)R^(c), —O(CH₂)_(u)C(O)R^(c),        —(CH₂)_(k)CONR^(d)R^(d), —(CH₂)_(k)NR^(d)C(O)R^(c),        —NR^(d)(CH₂)_(u)CONR^(d)R^(d), —NR^(d)(CH₂)_(u)NR^(d)C(O)R^(c),        —O(CH₂)_(u)CONR^(d)R^(d), —O(CH₂)_(u)NR^(d)C(O)R^(c),        —(CH₂)_(k)S(O)₂NR^(d)R^(d), —(CH₂)_(k)NR^(d)S(O)₂R^(c),        —(CH₂)_(k)S(O)₂R^(c), —(CH₂)_(k)S(O)R^(c), —(CH₂)_(k)SR^(d),        —NR^(d)(CH₂)_(u)S(O)₂NR^(d)R^(d),        —NR^(d)(CH₂)_(u)NR^(d)S(O)₂R^(c), —NR^(d)(CH₂)_(u)S(O)₂R^(c),        —NR^(d)(CH₂)_(u)S(O)R^(c), —NR^(d)(CH₂)_(u)SR^(d),        —O(CH₂)_(u)S(O)₂NR^(d)R^(d), —O(CH₂)_(u)NR^(d)S(O)₂R^(c),        —O(CH₂)_(u)S(O)₂R^(c), —O(CH₂)_(u)S(O)R^(c), and        —O(CH₂)_(u)SR^(c), wherein:    -   each R^(a) is independently selected from C₁₋₄ alkyl and C₁₋₄        haloalkyl,    -   each R^(b) is independently selected from C₃₋₆ cycloalkyl, C₃₋₆        halocycloalkyl, C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(c) is independently selected from —OH, C₁₋₈ alkyl, C₁₋₈        haloalkyl, C₃₋₈ cycloalkyl, C₃₋₈ halocycloalkyl, C₆₋₁₀ aryl,        (C₆₋₁₀ aryl)-(C₁₋₈ alkyl), 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(d) is independently selected from hydrogen and C₁₋₈        alkyl,    -   each subscript k is independently selected from 0, 1, 2, 3, 4,        5, and 6, and    -   each subscript u is independently selected from 1, 2, 3, 4, 5,        and 6; and    -   R⁴ is selected from hydrogen, halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy,        C₁₋₄ haloalkyl, and C₁₋₄ haloalkoxy.

In some embodiments, the disease or condition associated with P.gingivalis infection is a brain disorder selected from Alzheimer'sdisease, Down's syndrome, epilepsy, autism, Parkinson's disease,essential tremor, fronto-temporal dementia, progressive supranuclearpalsy, amyotrophic lateral sclerosis, Huntington's disease, multiplesclerosis, mild cognitive impairment, age associated memory impairment,chronic traumatic encephalopathy, stroke, Lewy Body disease, multiplesystem atrophy, schizophrenia, and depression. In some embodiments, thedisease or condition associated with P. gingivalis infection isAlzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(R)-lysinyl)-benzothiazole;compound 3.

FIG. 1B shows the structure of2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(R)-lysinyl)-benzothiazole;compound 4.

FIG. 1C shows that compound 4 prevents gingipain-induced death ofdifferentiated SHSY-5Y cells, but compound 3 does not. Compound 3exhibits a Kgp IC₅₀ between 10 and 25 nM, and compound 4 exhibits a KgpIC₅₀ between 1 and 10 nM.

FIG. 2A shows the structure of compound 1.

FIG. 2B shows the structure of compound 2.

FIG. 2C shows that either compound 1 or compound 2 preventsgingipain-induced death of differentiated SHSY-5Y cells, but thatcompound 2 is more effective. Compound 2 a Kgp IC₅₀ between 1 and 10 nMand a trypsin IC₅₀ of 5,000 nM.

FIG. 3A shows the structure of Compound 43.

FIG. 3B shows that Compound 43, an irreversible covalent lysinegingipain inhibitor having a sub-nanomolar IC₅₀ value, protects SHSYSYcells from P. gingivalis-induced toxicity in vitro compared to theantibiotic moxifloxacin.

FIG. 4 shows that intrahippocampal injection of gingipains into mousebrain causes neurodegeneration after 7 days.

FIG. 5 shows that RgpB brain infiltration overlaps withneurodegeneration of the subgranular zone in the hippocampus of BalbCmice infected with P. gingivalis orally for 6 weeks.

FIG. 6A shows that wild-type mice infected with P. gingivalis showcognitive impairment on the Novel Object Recognition task at the 6 weektime point. Infected mice spend equal amounts of time exploring a noveland familiar object, while normal mice spend increased time on the novelobject.

FIG. 6B shows the discrimination index(T_(Novel)−T_(familiar))/T_(total) for the uninfected mice and theinfected mice.

FIG. 7 shows brain levels of abeta 42 (“Ab42”) measured with ELISA,indicating that infection increases brain abeta and this can be resolvedby treatment with compound 2 mid-way through the infection.Additionally, the rise in abeta 42 is prevented when the bacteria usedfor infection does not express Kgp.

FIG. 8 shows that aged dogs with cognitive impairment are stronglypositive for brain Kgp.

FIG. 9 shows phamacokinetic data for a Kgp inhibitor, Kyt-36,demonstrating that ritonavir (RTC) increases the half-life of orallyadministered Kyt-36.

FIG. 10 shows an example of a “click chemistry” compound that can beused to create radiolabeled PET/SPECT imaging agents or capture agentsfor in vitro assays or diagnostics. In FIG. 10, R represents aradionuclide or a radionuclide-substituted moiety (e.g.,R=¹⁸F-alkylene).

DETAILED DESCRIPTION OF THE INVENTION I. General

Inhibition of Kgp with a variety of inhibitors has been shown to protectcells, prevent bacterial growth, increase immune system surveillance ofthe bacteria, and protect against reinfection. The present inventionprovides potent and selective nonpeptidic compounds with improvedproperties over previously described compounds. As demonstrated herein,Kgp inhibitors of the invention can prevent cell death caused bygingipains or P. gingivalis in an SH-SYSY cell model. The compounds canbe used to prevent cell death, inflammation, and other pathology in avariety of diseases associated with P. gingivalis infection, includingaging-related conditions such as Alzheimer's disease.

II. Definitions

As used herein, the term “alkyl,” by itself or as part of anothersubstituent, refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated. Alkyl can includeany number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 20 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted. “Substituted alkyl” groups can be substituted with one ormore groups selected from halo, hydroxy, amino, alkylamino, amido, acyl,nitro, cyano, and alkoxy.

As used herein, the term “alkoxy,” by itself or as part of anothersubstituent, refers to a group having the formula —OR, wherein R isalkyl. The term “lower alkoxyl” refers to an alkoxy radical having fromone to seven carbons, e.g., methoxyl, ethoxyl, propoxyl, butoxyl,pentoxyl, hexoxyl, or heptoxyl radical.

As used herein, the term “cycloalkyl,” by itself or as part of anothersubstituent, refers to a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Cycloalkyl can includeany number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈,C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂. Saturated monocyclic cycloalkyl ringsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl ringsinclude, for example, norbornane, [2.2.2] bicyclooctane,decahydronaphthalene and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. When cycloalkyl is a saturated monocyclic C₃₋₈cycloalkyl, exemplary groups include, but are not limited tocyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl. When cycloalkyl is a saturated monocyclic C₃₋₆ cycloalkyl,exemplary groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can besubstituted or unsubstituted. “Substituted cycloalkyl” groups can besubstituted with one or more groups selected from halo, hydroxy, amino,alkylamino, amido, acyl, nitro, cyano, and alkoxy. The term “lowercycloalkyl” refers to a cycloalkyl radical having from three to sevencarbons including, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cycloheptyl.

As used herein, the term “alkylene” refers to an alkyl group, as definedabove, linking at least two other groups (i.e., a divalent alkylradical). The two moieties linked to the alkylene group can be linked tothe same carbon atom or different carbon atoms of the alkylene group.

As used herein, the term “heteroalkyl,” by itself or as part of anothersubstituent, refers to an alkyl group of any suitable length and havingfrom 1 to 3 heteroatoms such as N, O and S. For example, heteroalkyl caninclude ethers, thioethers and alkyl-amines. Additional heteroatoms canalso be useful, including, but not limited to, B, Al, Si and P. Theheteroatoms can be oxidized to form moieties such as, but not limitedto, —S(O)— and —S(O)₂—. The heteroatom portion of the heteroalkyl canreplace a hydrogen of the alkyl group to form a hydroxy, thio or aminogroup. Alternatively, the heteroartom portion can be the connectingatom, or be inserted between two carbon atoms.

As used herein, the term “heteroalkylene” refers to a heteroalkyl group,as defined above, linking at least two other groups (i.e., a divalentheteroalkyl radical). The two moieties linked to the heteroalkylenegroup can be linked to the same atom or different atoms of theheteroalkylene group.

As used herein, the terms “halo” and “halogen,” by themselves or as partof another substituent, refer to a fluorine, chlorine, bromine, oriodine atom.

As used herein, the term “haloalkyl,” by itself or as part of anothersubstituent, refers to an alkyl group where some or all of the hydrogenatoms are replaced with halogen atoms. As for alkyl groups, haloalkylgroups can have any suitable number of carbon atoms, such as C₁₋₆. Forexample, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In someinstances, the term “perfluoro” can be used to define a compound orradical where all the hydrogens are replaced with fluorine. For example,perfluoromethyl refers to 1,1,1-trifluoromethyl.

As used herein, the term “haloalkoxy,” by itself or as part of anothersubstituent, refers to an alkoxy group where some or all of the hydrogenatoms are replaced with halogen atoms.

As used herein, the term “halocycloalkyl,” by itself or as part ofanother substituent, refers to a cycloalkyl group where some or all ofthe hydrogen atoms are replaced with halogen atoms.

As used herein, the term “aryl,” by itself or as part of anothersubstituent, refers to an aromatic ring system having any suitablenumber of ring atoms and any suitable number of rings. Aryl groups caninclude any suitable number of ring atoms, such as 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6to 14 ring members. Aryl groups can be monocyclic, fused to formbicyclic (e.g., benzocyclohexyl) or tricyclic groups, or linked by abond to form a biaryl group. Representative aryl groups include phenyl,naphthyl and biphenyl. Other aryl groups include benzyl, having amethylene linking group. Some aryl groups have from 6 to 12 ringmembers, such as phenyl, naphthyl or biphenyl. Other aryl groups havefrom 6 to 10 ring members, such as phenyl or naphthyl. Some other arylgroups have 6 ring members, such as phenyl. Aryl groups can besubstituted or unsubstituted. “Substituted aryl” groups can besubstituted with one or more groups selected from halo, hydroxy, amino,alkylamino, amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “heteroaryl,” by itself or as part of anothersubstituent, refers to a monocyclic or fused bicyclic or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5of the ring atoms are a heteroatom such as N, O or S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heteroaryl groups can include any numberof ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitablenumber of heteroatoms can be included in the heteroaryl groups, such as1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2to 5, 3 to 4, or 3 to 5. Heteroaryl groups can have from 5 to 8 ringmembers and from 1 to 4 heteroatoms, or from 5 to 8 ring members andfrom 1 to 3 heteroatoms, or from 5 to 6 ring members and from 1 to 4heteroatoms, or from 5 to 6 ring members and from 1 to 3 heteroatoms.The heteroaryl group can include groups such as pyrrole, pyridine,imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, and isoxazole. The heteroarylgroups can also be fused to aromatic ring systems, such as a phenylring, to form members including, but not limited to, benzopyrroles suchas indole and isoindole, benzopyridines such as quinoline andisoquinoline, benzopyrazine (quinoxaline), benzopyrimidine(quinazoline), benzopyridazines such as phthalazine and cinnoline,benzothiophene, and benzofuran. Other heteroaryl groups includeheteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groupscan be substituted or unsubstituted. “Substituted heteroaryl” groups canbe substituted with one or more groups selected from halo, hydroxy,amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

The heteroaryl groups can be linked via any position on the ring. Forexample, pyrrole includes 1-, 2- and 3-pyrrole, pyridine includes 2-, 3-and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazoleincludes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes2-, 4-, 5- and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine,1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-,5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiopheneincludes 2- and 3-thiophene, furan includes 2- and 3-furan, thiazoleincludes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and5-isothiazole, oxazole includes 2-, 4- and 5-oxazole, isoxazole includes3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindoleincludes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline,isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2-and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline,benzothiophene includes 2- and 3-benzothiophene, and benzofuran includes2- and 3-benzofuran.

Some heteroaryl groups include those having from 5 to 10 ring membersand from 1 to 3 ring atoms including N, O or S, such as pyrrole,pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, and benzofuran. Other heteroaryl groupsinclude those having from 5 to 8 ring members and from 1 to 3heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole,pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, andisoxazole. Some other heteroaryl groups include those having from 9 to12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, benzofuran and bipyridine. Still otherheteroaryl groups include those having from 5 to 6 ring members and from1 to 2 ring atoms including N, O or S, such as pyrrole, pyridine,imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan,thiazole, isothiazole, oxazole, and isoxazole.

Some heteroaryl groups include from 5 to 10 ring members and onlynitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline,quinazoline, phthalazine, and cinnoline. Other heteroaryl groups includefrom 5 to 10 ring members and only oxygen heteroatoms, such as furan andbenzofuran. Some other heteroaryl groups include from 5 to 10 ringmembers and only sulfur heteroatoms, such as thiophene andbenzothiophene. Still other heteroaryl groups include from 5 to 10 ringmembers and at least two heteroatoms, such as imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline,quinazoline, phthalazine, and cinnoline.

As used herein the term “heterocyclyl,” by itself or as part of anothersubstituent, refers to a saturated ring system having from 3 to 12 ringmembers and from 1 to 4 heteroatoms of N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heterocyclyl groups can include anynumber of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Anysuitable number of heteroatoms can be included in the heterocyclylgroups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to4, or 3 to 4. The heterocyclyl group can include groups such asaziridine, azetidine, pyrrolidine, piperidine, azepane, azocane,quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane(tetrahydropyran), oxepane, thiirane, thietane, thiolane(tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane,morpholine, thiomorpholine, dioxane, or dithiane. The heterocyclylgroups can also be fused to aromatic or non-aromatic ring systems toform members including, but not limited to, indoline. Heterocyclylgroups can be unsubstituted or substituted. “Substituted heterocyclyl”groups can be substituted with one or more groups selected from halo,hydroxy, amino, oxo (═O), alkylamino, amido, acyl, nitro, cyano, andalkoxy.

The heterocyclyl groups can be linked via any position on the ring. Forexample, aziridine can be 1- or 2-aziridine, azetidine can be 1- or2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine canbe 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine,piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1-or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine,isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

When heterocyclyl includes 3 to 8 ring members and 1 to 3 heteroatoms,representative members include, but are not limited to, pyrrolidine,piperidine, tetrahydrofuran, oxane, tetrahydrothiophene, thiane,pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxzoalidine,thiazolidine, isothiazolidine, morpholine, thiomorpholine, dioxane anddithiane. Heterocyclyl can also form a ring having 5 to 6 ring membersand 1 to 2 heteroatoms, with representative members including, but notlimited to, pyrrolidine, piperidine, tetrahydrofuran,tetrahydrothiophene, pyrazolidine, imidazolidine, piperazine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, andmorpholine.

As used herein, the term “thiol-reactive group” refers to a functionalgroup capable of forming a reversible or irreversible covalent bond witha thiol group (i.e., a group having the structure “—SH”) such as thethiol group present in the α-sidechain of cysteine. Non-limitingexamples of thiol reactive groups include thiazol-2-yl-carbonyl;benzothiazol-2-yl-carbonyl; oxazol-2-yl-carbonyl;benzooxazol-2-yl-carbonyl; pyridin-2-yl-carbonyl;pyrimidin-4-yl-carbonyl; pyrimidin-2-yl-carbonyl;isoxazol-5-yl-carbonyl; isoxazol-3-yl-carbonyl;1,2,4-oxadiazol-3-yl-carbonyl; 1,2,4-oxadiazol-5-yl-carbonyl;maleimidyl; pyridinyldisulfanyl (including pyridin-2-yldisulfanyl);cyano; ethynyl; fluoromethyl-carbonyl; acyloxymethyl-carbonyl;aryloxymethyl-carbonyl; alkylsulfonyl-vinyl; and arylsulfonyl-vinyl.Other thiol-reactive groups are known to those of skill in the artincluding, for example, those described by Hermanson (BioconjugateTechniques, 3^(rd) Ed. 2013, Academic Press, San Diego).

A “masked thiol-reactive group” refers to a non-reactive precursormoiety that can be converted into a functional group capable of forminga reversible or irreversible covalent bond with a thiol group.

As used herein, the term “amine protecting group” refers to a chemicalmoiety that renders an amino group unreactive, but is also removable soas to restore the amino group. Examples of amine protecting groupsinclude, but are not limited to, benzyloxycarbonyl,9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc),allyloxycarbonyl (Alloc), acetamido, phthalimido, and the like. Otheramine protecting groups are known to those of skill in the artincluding, for example, those described by Green and Wuts (ProtectiveGroups in Organic Synthesis, 4^(th) Ed. 2007, Wiley-Interscience, NewYork).

As used herein, the term “carbonyl,” by itself or as part of anothersubstituent, refers to —C(O)—, i.e., a carbon atom double-bonded tooxygen and bound to two other groups in the moiety having the carbonyl.

As used herein, the term “amino” refers to a moiety —NR₃, wherein each Rgroup is H or alkyl. An amino moiety can be ionized to form thecorresponding ammonium cation.

As used herein, the term “hydroxy” refers to the moiety —OH.

As used herein, the term “cyano” refers to a carbon atom triple-bondedto a nitrogen atom (i.e., the moiety —C≡N).

As used herein, the term “carboxy” refers to the moiety —C(O)OH. Acarboxy moiety can be ionized to form the corresponding carboxylateanion.

As used herein, the term “amido” refers to a moiety —NRC(O)R or—C(O)NR₂, wherein each R group is H or alkyl.

As used herein, the term “nitro” refers to the moiety —NO₂.

As used herein, the term “oxo” refers to an oxygen atom that isdouble-bonded to a compound (i.e., O═).

As used herein, the term “pharmaceutically acceptable excipient” refersto a substance that aids the administration of an active agent to asubject. By “pharmaceutically acceptable,” it is meant that theexcipient is compatible with the other ingredients of the formulationand is not deleterious to the recipient thereof. Pharmaceuticalexcipients useful in the present invention include, but are not limitedto, binders, fillers, disintegrants, lubricants, glidants, coatings,sweeteners, flavors and colors.

As used herein, the term “salt” refers to acid or base salts of thecompounds of the invention. Illustrative examples of pharmaceuticallyacceptable salts are mineral acid (hydrochloric acid, hydrobromic acid,phosphoric acid, and the like) salts, organic acid (acetic acid,propionic acid, glutamic acid, citric acid and the like) salts,quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.It is understood that the pharmaceutically acceptable salts arenon-toxic.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds can be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

As used herein, the terms “Porphyromonas gingivalis” and “P. gingivalis”refer to the gram-negative asaccharolytic bacterium that is recognizedas a key causative microbe in the pathogenesis of periodontitis andrelated conditions. “P. gingivalis infection” refers to the invasion andcolonization of P. gingivalis in a bodily tissue such as the gums or thebrain. P. gingivalis infection is frequently characterized by subsequenttissue injury and disease.

As used herein, the term “gingipain” refers to cysteine proteasesexpressed by P. gingivalis having trypsin-like specificity (i.e.,Lys-Xaa and Arg-Xaa). Gingipains are recognized as the major virulencefactors of the P. gingivalis and contribute to bacterial attachment andcolonization, nutrient acquisition, evasion of host defenses, and tissueinvasion. The terms “lysine gingipain” and “Kgp” are usedinterchangeably to refer to the P. gingivalis lysine-specific gingipainknown by EC number EC 3.4.22.47.

As used herein, the terms “treat,” “treatment,” and “treating” refer toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., cognitive impairment), includingany objective or subjective parameter such as abatement; remission;diminishing of symptoms or making the symptom, injury, pathology orcondition more tolerable to the patient; decreasing the frequency orduration of the symptom or condition; or, in some situations, preventingthe onset of the symptom. The treatment or amelioration of symptoms canbe based on any objective or subjective parameter; including, e.g., theresult of a physical examination.

As used herein the terms “effective amount” and “therapeuticallyeffective amount” refer to a dose of a compound such as a Kgp inhibitorthat produces therapeutic effects for which it is administered. Theexact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see,e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,The Art, Science and Technology of Pharmaceutical Compounding (1999);Pickar, Dosage Calculations (1999); Goodman & Gilman's ThePharmacological Basis of Therapeutics, 11^(th) Edition, 2006, Brunton,Ed., McGraw-Hill; and Remington: The Science and Practice of Pharmacy,21^(st) Edition, 2005, Hendrickson, Ed., Lippincott, Williams &Wilkins).

As used herein, the term “Alzheimer's disease” refers to a progressivedisease of the central nervous system in humans and other mammals. It ismanifested by dementia (especially in the elderly); disorientation; lossof memory; difficulty with language, calculation, or visual-spatialskills; and psychiatric manifestations. Alzheimer's disease isassociated with progressive neurodegeneration and characteristicpathology, namely beta amyloid plaques and tau tangles.

As used herein, the term “subject” refers to animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like.

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding that explicit value. If “X”were the value, “about X” or “around X” would indicate a value from 0.9Xto 1.1X, and more preferably, a value from 0.95X to 1.05X. Any referenceto “about X” or “around X” specifically indicates at least the values X,0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and1.05X. Thus, “about X” and “around X” are intended to teach and providewritten description support for a claim limitation of, e.g., “0.98X.”

III. Inhibitors of Lysine Gingipain

In one aspect, the invention provides a compound according to Formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Z is a thiol-reactive group or a masked thiol-reactive group;    -   A is selected from —CH₂— and —O—;    -   B and D are independently selected from hydrogen, halogen, C₁₋₄        haloalkyl, and C₁₋₄ haloalkoxy;    -   R¹ is selected from hydrogen and an amine protecting group;    -   R² is hydrogen; and    -   R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₁₋₈ alkyl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, -L-R⁵, and —OR⁶, wherein    -   L is selected from —O—, —NR—, C₁₋₄ alkylene, and 2- to        4-membered heteroalkylene, wherein R is selected from hydrogen        and C₁₋₈ alkyl,    -   R⁵ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₃₋₈ cycloalkyl, and 5-to-12 membered saturated heterocyclyl,        and    -   —OR⁶ and the carbonyl to which it is bonded form an amine        protecting group,    -   and wherein R³ is optionally substituted with one or more        substituents selected from halo, —CN, —NO₂, —N₃, —OH, R^(a),        R^(b), —OR^(a), —OR^(b), —(CH₂)_(k)C(O)R^(c),        —NR^(d)(CH₂)_(u)C(O)R^(c), —O(CH₂)_(u)C(O)R^(c),        —(CH₂)_(k)CONR^(d)R^(d), —(CH₂)_(k)NR^(d)C(O)R^(c),        —NR^(d)(CH₂)_(u)CONR^(d)R^(d), —NR^(d)(CH₂)_(u)NR^(d)C(O)R^(c),        —O(CH₂)_(u)CONR^(d)R^(d), —O(CH₂)_(u)NR^(d)C(O)R^(c),        —(CH₂)_(k)S(O)₂NR^(d)R^(d), (CH₂)_(k)NR^(d)S(O)₂R^(c),        —(CH₂)_(k)S(O)₂R^(c), —(CH₂)_(k)S(O)R^(c), —(CH₂)_(k)SR^(d),        —NR^(d)(CH₂)_(u)S(O)₂NR^(d)R^(d),        —NR^(d)(CH₂)_(u)NR^(d)S(O)₂R^(c), —NR^(d)(CH₂)_(u)S(O)₂R^(c),        —NR^(d)(CH₂)_(u)S(O)R^(c), —NR^(d)(CH₂)_(u)SR^(d),        —O(CH₂)_(u)S(O)₂NR^(d)R^(d), —O(CH₂)_(u)NR^(d)S(O)₂R^(c),        —O(CH₂)_(u)S(O)₂R^(c), —O(CH₂)_(u)S(O)R^(c), and        —O(CH₂)_(u)SR^(c), wherein:    -   each R^(a) is independently selected from C₁₋₄ alkyl and C₁₋₄        haloalkyl,    -   each R^(b) is independently selected from C₃₋₆ cycloalkyl, C₃₋₆        halocycloalkyl, C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(c) is independently selected from —OH, C₁₋₈ alkyl, C₁₋₈        haloalkyl, C₃₋₈ cycloalkyl, C₃₋₈ halocycloalkyl, C₆₋₁₀ aryl,        (C₆₋₁₀ aryl)-(C₁₋₈ alkyl), 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(d) is independently selected from hydrogen and C₁₋₈        alkyl,    -   each subscript k is independently selected from 0, 1, 2, 3, 4,        5, and 6, and    -   each subscript u is independently selected from 1, 2, 3, 4, 5,        and 6; and    -   R⁴ is selected from hydrogen, halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy,        C₁₋₄ haloalkyl, and C₁₋₄ haloalkoxy;    -   provided that when Z is benzothiazol-2-yl-carbonyl, A is —CH₂—,        and B, D, and R¹ are hydrogen, then R³ is other than benzyloxy,        substituted benzyloxy, or 1-(3-phenyl-propanoyl)piperidin-3-yl,        and    -   provided that when Z is phenoxymethyl-carbonyl or substituted        phenoxymethyl-carbonyl, A is —CH₂—, and B and D are hydrogen,        then R³ is other than (2-phenyl)ethyl or substituted        (2-phenyl)ethyl.

In some embodiments, the compound of Formula I has a structure accordingto Formula Ia:

In some embodiments, the compound of Formula I has a structure accordingto Formula Ib:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   B and D are independently selected from hydrogen, halogen,        halomethyl, and halomethoxy.

In some embodiments, the invention provides compounds of Formula I,Formula Ia, or Formula Ib, and pharmaceutically acceptable saltsthereof, wherein Z is selected from benzothiazol-2-yl-carbonyl;thiazol-2-yl-carbonyl; oxazol-2-yl-carbonyl; benzooxazol-2-yl-carbonyl;pyridin-2-yl-carbonyl; pyrimidin-4-yl-carbonyl; pyrimidin-2-yl-carbonyl;isoxazol-5-yl-carbonyl; isoxazol-3-yl-carbonyl;1,2,4-oxadiazol-3-yl-carbonyl; 1,2,4-oxadiazol-5-yl-carbonyl; cyano;ethynyl; fluoromethyl-carbonyl; acyloxymethyl-carbonyl;aryloxymethyl-carbonyl; alkylsulfonyl-vinyl; and arylsulfonyl-vinyl;each of which is optionally substituted with one or more substituentsselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkyl, C₁₋₄ haloalkoxy,halogen, and —N₃.

In some embodiments, Z is selected from benzothiazol-2-yl-carbonyl,halogen-substituted aryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl.

In some embodiments, the invention provides compounds of Formula I orFormula Ia as described above, and pharmaceutically acceptable saltsthereof, wherein Z is selected from benzothiazol-2-yl-carbonyl;thiazol-2-yl-carbonyl; oxazol-2-yl-carbonyl; benzooxazol-2-yl-carbonyl;pyridin-2-yl-carbonyl; pyrimidin-4-yl-carbonyl; pyrimidin-2-yl-carbonyl;isoxazol-5-yl-carbonyl; isoxazol-3-yl-carbonyl;1,2,4-oxadiazol-3-yl-carbonyl; 1,2,4-oxadiazol-5-yl-carbonyl; cyano;ethynyl; fluoromethyl-carbonyl; acyloxymethyl-carbonyl;aryloxymethyl-carbonyl; alkylsulfonyl-vinyl; and arylsulfonyl-vinyl;wherein Z is optionally substituted with one or more substituentsselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkyl, C₁₋₄ haloalkoxy,halogen, and —N₃; and wherein R³ is selected from C₆₋₁₀ aryl, 5-to-12membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturatedheterocyclyl, and -L-R⁵. In some such embodiments, R¹ and R² are H; Band D are independently selected from hydrogen and fluoro; A is —CH₂—;and R⁴ is selected from hydrogen and C₁₋₄ alkyl.

In some embodiments, the invention provides compounds of Formula Ib asdescribed above, and pharmaceutically acceptable salts thereof, whereinZ is selected from benzothiazol-2-yl-carbonyl; thiazol-2-yl-carbonyl;oxazol-2-yl-carbonyl; benzooxazol-2-yl-carbonyl; pyridin-2-yl-carbonyl;pyrimidin-4-yl-carbonyl; pyrimidin-2-yl-carbonyl;isoxazol-5-yl-carbonyl; isoxazol-3-yl-carbonyl;1,2,4-oxadiazol-3-yl-carbonyl; 1,2,4-oxadiazol-5-yl-carbonyl; cyano;ethynyl; fluoromethyl-carbonyl; acyloxymethyl-carbonyl;aryloxymethyl-carbonyl; alkylsulfonyl-vinyl; and arylsulfonyl-vinyl;wherein Z is optionally substituted with one or more substituentsselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkyl, C₁₋₄ haloalkoxy,halogen, and —N₃; and wherein R³ is selected from C₆₋₁₀ aryl, 5-to-12membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturatedheterocyclyl, and -L-R⁵. In some such embodiments, R¹ and R² are H; Band D are independently selected from hydrogen and fluoro; and A is—CH₂—.

In some embodiments, the invention provides compounds of Formula I orFormula Ia as described above, and pharmaceutically acceptable saltsthereof, wherein Z is selected from benzothiazol-2-yl-carbonyl,halogen-substituted aryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl; and wherein R³ is selected from C₆₋₁₀ aryl,5-to-12 membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturatedheterocyclyl, and -L-R⁵. In some such embodiments, R¹ and R² are H; Band D are independently selected from hydrogen and fluoro; A is —CH₂—;and R⁴ is selected from hydrogen and C₁₋₄ alkyl.

In some embodiments, the invention provides compounds of Formula Ib asdescribed above, and pharmaceutically acceptable salts thereof, whereinZ is selected from benzothiazol-2-yl-carbonyl, halogen-substitutedaryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl; and wherein R³ is selected from C₆₋₁₀ aryl,5-to-12 membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturatedheterocyclyl, and -L-R⁵. In some such embodiments, R¹ and R² are H; Band D are independently selected from hydrogen and fluoro; and A is—CH₂—.

In some embodiments, the invention provides compounds of Formula I orFormula Ia as described above, and pharmaceutically acceptable saltsthereof, wherein Z is selected from benzothiazol-2-yl-carbonyl;thiazol-2-yl-carbonyl; oxazol-2-yl-carbonyl; benzooxazol-2-yl-carbonyl;pyridin-2-yl-carbonyl; pyrimidin-4-yl-carbonyl; pyrimidin-2-yl-carbonyl;isoxazol-5-yl-carbonyl; isoxazol-3-yl-carbonyl;1,2,4-oxadiazol-3-yl-carbonyl; oxadiazol-5-yl-carbonyl; cyano; ethynyl;fluoromethyl-carbonyl; acyloxymethyl-carbonyl; aryloxymethyl-carbonyl;alkylsulfonyl-vinyl; and arylsulfonyl-vinyl; wherein Z is optionallysubstituted with one or more substituents selected from C₁₋₄ alkyl, C₁₋₄alkoxy, C₁₋₄ haloalkyl, C₁₋₄ haloalkoxy, halogen, and —N₃; and whereinR³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, C₃₋₈cycloalkyl, 5-to-12 membered saturated heterocyclyl, and -L-R⁵. In somesuch embodiments, R¹ and R² are H; B and D are independently selectedfrom hydrogen and fluoro; and A is —CH₂—. In some such embodiments, R⁴is selected from hydrogen and methyl.

In some embodiments, the invention provides compounds of Formula I orFormula Ia as described above, and pharmaceutically acceptable saltsthereof, wherein Z is selected from benzothiazol-2-yl-carbonyl,halogen-substituted aryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl; and wherein R³ is selected from C₆₋₁₀ aryl,5-to-12 membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturatedheterocyclyl, and -L-R⁵. In some such embodiments, R¹ and R² are H; Band D are independently selected from hydrogen and fluoro; and A is—CH₂—. In some such embodiments, R⁴ is selected from hydrogen andmethyl.

In some embodiments, the invention provides compounds of Formula I orFormula Ia as described above, and pharmaceutically acceptable saltsthereof, wherein:

A is —CH₂—;

B and D are hydrogen;

Z is selected from benzothiazol-2-yl-carbonyl, halogen-substitutedaryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl;

R¹ and R² are H; and

R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, C₃₋₈cycloalkyl, 5-to-12 membered saturated heterocyclyl, and -L-R⁵. In somesuch embodiments, R⁴ is hydrogen or methyl. In some such embodiments, R⁴is hydrogen.

In some embodiments, the invention provides compounds of Formula Ib asdescribed above, and pharmaceutically acceptable salts thereof, wherein:

A is —CH₂—;

B and D are hydrogen;

Z is selected from benzothiazol-2-yl-carbonyl, halogen-substitutedaryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl;

R¹ and R² are H; and

R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, C₃₋₈cycloalkyl, 5-to-12 membered saturated heterocyclyl, and -L-R⁵.

In some embodiments, the invention provides compounds of Formula I orFormula Ia as described above, and pharmaceutically acceptable saltsthereof, wherein:

A is —CH₂—;

B is hydrogen;

D is fluoro;

Z is selected from benzothiazol-2-yl-carbonyl, halogen-substitutedaryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl;

R¹ and R² are H; and

R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, C₃₋₈cycloalkyl, 5-to-12 membered saturated heterocyclyl, and -L-R⁵. In somesuch embodiments, R⁴ is hydrogen or methyl. In some such embodiments, R⁴is hydrogen.

In some embodiments, the invention provides compounds of Formula Ib asdescribed above, and pharmaceutically acceptable salts thereof, wherein:

A is —CH₂—;

B is hydrogen;

D is fluoro;

Z is selected from benzothiazol-2-yl-carbonyl, halogen-substitutedaryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl;

R¹ and R² are H; and

R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, C₃₋₈cycloalkyl, 5-to-12 membered saturated heterocyclyl, and -L-R⁵.

In some embodiments, the invention provides compounds of Formula I orFormula Ia as described above, and pharmaceutically acceptable saltsthereof, wherein:

A is —CH₂—;

B is hydrogen;

D is hydrogen or fluoro;

Z is benzothiazol-2-yl-carbonyl or halogen-substitutedaryloxymethyl-carbonyl;

R¹ and R² are H;

R³ is selected from cyclohexyl, cyclopentyl, morpholino, phenyl,piperidinyl, pyridinyl, tetrahydrofuranyl, tetrahydropyranyl,1,2,3,4-tetrahydronaphthyl, and thiazolyl,

each of which is optionally substituted with 1-3 members selected fromthe group consisting of methyl, methoxy, trifluoromethyl, acetyl, and—N₃; and

R⁴ is hydrogen or methyl. In some such embodiments, R⁴ is hydrogen. Insome such embodiments, D and R⁴ are hydrogen. In some such embodiments,D and R⁴ are hydrogen, and Z is(2,3,5,6-tetrafluorophenoxy)methyl-carbonyl.

In some embodiments, the invention provides compounds of Formula Ib asdescribed above, and pharmaceutically acceptable salts thereof, wherein:

A is —CH₂—;

B is hydrogen;

D is hydrogen or fluoro;

Z is benzothiazol-2-yl-carbonyl or halogen-substitutedaryloxymethyl-carbonyl;

R¹ and R² are H; and

R³ is selected from cyclohexyl, cyclopentyl, morpholino, phenyl,piperidinyl, pyridinyl, tetrahydrofuranyl, tetrahydropyranyl,1,2,3,4-tetrahydronaphthyl, and thiazolyl,

each of which is optionally substituted with 1-3 members selected fromthe group consisting of methyl, methoxy, trifluoromethyl, acetyl, and—N₃. In some such embodiments, D is hydrogen. In some such embodiments,D is hydrogen and Z is (2,3,5,6-tetrafluorophenoxy)methyl-carbonyl.

In some embodiments, the compound of Formula I has a structure accordingto Formula Ic:

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered        heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, and -L-R⁵,    -   wherein L is C₁₋₄ alkylene.

In some embodiments, the invention provides a compound of Formula Ic, ora pharmaceutically acceptable salt thereof, wherein R³ is selected fromcyclohexyl, cyclopentyl, morpholino, phenyl, piperidinyl, pyridinyl,tetrahydrofuranyl, tetrahydropyranyl, 1,2,3,4-tetrahydronaphthyl, andthiazolyl, each of which is optionally substituted with 1-3 membersselected from methyl, methoxy, trifluoromethyl, acetyl, and —N₃.

In some embodiments, the compound of Formula I, Formula Ia, Formula Ib,or Formula Ic is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula I, Formula Ia, Formula Ib,or Formula Ic is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the invention provides compounds of Formula I,Formula Ia, or Formula Ib wherein A is —O—, including compoundsaccording to Formula C1:

In some embodiments, the invention provides compounds of Formula I,Formula Ia, or Formula Ib wherein B is halo; D is halo; or B and D arehalo; including compounds according to Formula C2, Formula C3, andFormula C4:

In some embodiments, the invention provides compounds of Formula I,Formula Ia, or Formula Ib wherein Z is selected fromthiazol-2-yl-carbonyl; oxazol-2-yl-carbonyl; benzooxazol-2-yl-carbonyl;pyridin-2-yl-carbonyl; pyrimidin-4-yl-carbonyl; pyrimidin-2-yl-carbonyl;isoxazol-5-yl-carbonyl; isoxazol-3-yl-carbonyl;1,2,4-oxadiazol-3-yl-carbonyl; 1,2,4-oxadiazol-5-yl-carbonyl;maleimidyl; pyridinyldisulfanyl (including pyridin-2-yldisulfanyl);cyano; ethynyl; fluoromethyl-carbonyl; acyloxymethyl-carbonyl;aryloxymethyl-carbonyl; alkylsulfonyl-vinyl; and arylsulfonyl-vinyl.

In some embodiments, the invention provides compounds of Formula I,Formula Ia, or Formula Ib wherein Z is selected fromthiazol-2-yl-carbonyl; pyridin-2-yl-carbonyl; cyano; ethynyl;fluoromethylcarbonyl; and 2,3,5,6-tetrafluorophenoxymethyl-carbonyl;including compounds according to Formula B1, Formula B2, Formula B3,Formula B4, Formula B5, and Formula B6:

In some embodiments, the invention provides a compound having astructure according to Formula Id:

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered        heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, and -L-R⁵,    -   wherein L is C₁₋₄ alkylene.

In some embodiments, the invention provides a compound of Formula Id, ora pharmaceutically acceptable salt thereof, wherein R³ is selected fromcyclohexyl, cyclopentyl, morpholino, phenyl, piperidinyl, pyridinyl,tetrahydrofuranyl, tetrahydropyranyl, 1,2,3,4-tetrahydronaphthyl, andthiazolyl, each of which is optionally substituted with 1-3 membersselected from methyl, methoxy, trifluoromethyl, acetyl, and —N₃. In somesuch embodiments, R³ is cyclopentyl.

In some embodiments, the compound of Formula Id is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula Id is selected from:

and pharmaceutically acceptable salts thereof

In some embodiments, the compound of Formula Id is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula I, Formula Ia, or FormulaIb is selected from

and pharmaceutically acceptable salts thereof.

In some embodiments, the invention provides compounds of Formula I,Formula Ia, or Formula Ib wherein Z is selected frompyridin-2-yl-carbonyl and thiazol-2-yl-carbonyl, and R³ is selected fromC₆₋₁₀ aryl and C₃₋₈ cycloalkyl. In some such embodiments, the compoundis selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the invention provides compounds of Formula I orFormula Ia, wherein R⁴ is selected from C₁₋₄ alkyl and C₁₋₄ haloalkyl.In some such embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a compound of Formula I,Formula Ia, Formula Ib, Formula Ic, or Formula Id, or a pharmaceuticallyacceptable salt thereof, wherein R³ is selected from cyclohexyl;1-methylcyclohexyl; 1-methoxycyclohexyl; cyclopentyl; morpholin-2-yl;4-acetylmorpholin-2-yl; phenyl; 2-trifluoromethylphenyl; 3-azidophenyl;piperidine-3-yl; 1-acetyl-piperidine-3-yl; pyridin-2-yl; pyridin-3-yl;pyridin-4-yl; 6-oxo-1,6-dihydropyridin-2-yl; tetrahydrofuran-2-yl;tetrahydro-2H-pyran-2-yl; tetrahydro-2H-pyran-3-yl;tetrahydro-2H-pyran-4-yl; 1,2,3,4-tetrahydronaphth-1-yl;1,2,3,4-tetrahydronaphth-2-yl; thiazol-5-yl; and thiazol-2-yl.

In some embodiments, the invention provides a compound of Formula I,Formula Ia, Formula Ib, Formula Ic, or Formula Id, or a pharmaceuticallyacceptable salt thereof, wherein R³ is selected from cyclohexyl;1-methylcyclohexyl; 1-methoxycyclohexyl; cyclopentyl;1,2,3,4-tetrahydronaphth-1-yl; and 1,2,3,4-tetrahydronaphth-2-yl; whichradicals are shown below. In some such embodiments, R³ is cyclopentyl.

In some embodiments, the invention provides a compound of Formula I,Formula Ia, Formula Ib, Formula Ic, or Formula Id, or a pharmaceuticallyacceptable salt thereof, wherein R³ is selected from morpholin-2-yl;4-acetylmorpholin-2-yl; piperidine-3-yl; 1-acetyl-piperidine-3-yl;tetrahydrofuran-2-yl; tetrahydro-2H-pyran-2-yl;tetrahydro-2H-pyran-3-yl; and tetrahydro-2H-pyran-4-yl; which radicalsare shown below.

In some embodiments, the invention provides a compound of Formula I,Formula Ia, Formula Ib, Formula Ic, or Formula Id, or a pharmaceuticallyacceptable salt thereof, wherein R³ is selected from phenyl;2-trifluoromethylphenyl; 3-azidophenyl; pyridin-2-yl; pyridin-3-yl;pyridin-4-yl; 6-oxo-1,6-dihydropyridin-2-yl; thiazol-5-yl; andthiazol-2-yl; which radicals are shown below.

The compounds described herein and methods of using them encompass thepreparation and use of therapeutically active enantiomers ordiastereoisomers of the described compounds. All such enantiomers anddiastereoisomers of these compounds are included in the scope of theinvention. Such compounds can be used as mixtures (e.g., racemicmixtures) or as isolated enantiomers or diastereoisomers.

Compounds of the invention can be prepared so as to includeradionuclides for use in diagnostic imaging application such as positronemission tomography (PET) and single-photon emission computed tomography(SPECT). For example, Kgp inhibitors as described herein can be preparedso as to include one or more radionuclides selected from oxygen-15(¹⁵O), nitrogen-13 (¹³N), carbon-11 (¹¹C) iodine-131 (¹³¹I), andfluorine-18 (¹⁸F). Such radiolabeled compounds can be used for PETimagining. Compounds of the invention can also be prepared in deuteratedform (i.e., having one or more deuterium atoms, ²H, in place of one morehydrogen atoms), tritiated form (i.e., having one or more tritium atoms,³H, in place of one more hydrogen atoms), or ¹⁴C-labeled form (i.e.,having one or more ¹⁴C atoms in place of one more carbon atoms).

In further embodiments, the invention provides compounds according toFormula Ie:

-   -   and pharmaceutically acceptable salts thereof, wherein    -   Z is a thiol-reactive group or a masked thiol-reactive group;    -   A is selected from —CH₂— and —O—;    -   B and D are independently selected from hydrogen, halogen, C₁₋₄        haloalkyl, and C₁₋₄ haloalkoxy;    -   R¹ is selected from hydrogen and an amine protecting group;    -   R² is hydrogen; and    -   R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₁₋₈ alkyl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, -L-R⁵, and —OR⁶, wherein    -   L is selected from —O—, —NR—, C₁₋₄ alkylene, and 2- to        4-membered heteroalkylene, wherein R is selected from hydrogen        and C₁₋₈ alkyl,    -   R⁵ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₃₋₈ cycloalkyl, and 5-to-12 membered saturated heterocyclyl,        and    -   —OR⁶ and the carbonyl to which it is bonded form an amine        protecting group,    -   and wherein R³ is optionally substituted with one or more        substituents selected from halo, —CN, —NO₂, —N₃, —OH, R^(a),        R^(b), —OR^(a), —OR^(b), —(CH₂)_(k)C(O)R^(c),        —NR^(d)(CH₂)_(u)C(O)R^(c), —O(CH₂)_(u)C(O)R^(c),        —(CH₂)_(k)CONR^(d)R^(d), —(CH₂)_(k)NR^(d)C(O)R^(c),        —NR^(d)(CH₂)_(u)CONR^(d)R^(d), —NR^(d)(CH₂)_(u)NR^(d)C(O)R^(c),        —O(CH₂)_(u)CONR^(d)R^(d), —O(CH₂)_(u)NR^(d)C(O)R^(c),        —(CH₂)_(k)S(O)₂NR^(d)R^(d), —(CH₂)_(k)NR^(d)S(O)₂R^(c),        —(CH₂)_(k)S(O)₂R^(c), —(CH₂)_(k)S(O)R^(c), —(CH₂)_(k)SR^(d),        —NR^(d)(CH₂)_(u)S(O)₂NR^(d)R^(d),        —NR^(d)(CH₂)_(u)NR^(d)S(O)₂R^(c), —NR^(d)(CH₂)_(u)S(O)₂R^(c),        —NR^(d)(CH₂)_(u)S(O)R^(c), —NR^(d)(CH₂)_(u)SR^(d),        —O(CH₂)_(u)S(O)₂NR^(d)R^(d), —O(CH₂)_(u)NR^(d)S(O)₂R^(c),        —O(CH₂)_(u)S(O)₂R^(c), —O(CH₂)_(u)S(O)R^(c), and        —O(CH₂)_(u)SR^(c), wherein:    -   each R^(a) is independently selected from C₁₋₄ alkyl and C₁₋₄        haloalkyl,    -   each R^(b) is independently selected from C₃₋₆ cycloalkyl, C₃₋₆        halocycloalkyl, C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(c) is independently selected from —OH, C₁₋₈ alkyl, C₁₋₈        haloalkyl, C₃₋₈ cycloalkyl, C₃₋₈ halocycloalkyl, C₆₋₁₀ aryl,        (C₆₋₁₀ aryl)-(C₁₋₈ alkyl), 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(d) is independently selected from hydrogen and C₁₋₈        alkyl,    -   each subscript k is independently selected from 0, 1, 2, 3, 4,        5, and 6, and    -   each subscript u is independently selected from 1, 2, 3, 4, 5,        and 6; and    -   R⁴ is selected from hydrogen, halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy,        C₁₋₄ haloalkyl, and C₁₋₄ haloalkoxy.

In some embodiments, the compound of Formula Ie has a structureaccording to Formula If:

It is to be understood that compounds of the invention do not include1-(3-phenylpropionyl)piperidine-3(R,S)-carboxylicacid-[4-amino-1(S)-(benzothiazole-2-carbonyl butyl]amide (i.e., A71561).

The compounds of the invention are highly active Kgp inhibitors,typically exhibiting Kgp Ki values and Kgp IC₅₀ values well below 1 μM.

The term “Ki” refers to inhibition constant. The Ki value for aparticular test compound can be measured as follows. Fifty microliters(μl) of an enzyme such as Kgp (1 nM in 50 mM bis-Tris propane [pH 8.0]containing 1% [vol/vol] Triton X-100 and 5 mM 2-mercaptoethanol) isadded to columns 1 to 11 of a 96-well plate, and 100 μl is added tocolumn 12. Two μl of the test compound (100 μl in 100% DMSO) is added tocolumn 12, and the sample is mixed three times by pipetting. Then, adoubling dilution is prepared across the plate by serial transfer intoadjacent wells. 50 μl of succinyl-Ala-Phe-Lys-(7-amido-4-methylcoumarin(“AMC;” 40 μM in buffer) is added to all wells, and the contents aremixed. The reaction is monitored for AMC fluorescence for 15 min at 25°C., and the progress curves are automatically converted to rates by theFluoroskan Ascent software.

The method can be used to assay enzymes including Kgp, RgpB, RgpA,trypsin, and cathepsin B. For RgpA and RgpB, the substrate can beZ-Arg-AMC. For trypsin, the buffer can contain 10 mM Tris and 10 mMCaCl₂) (pH 8.0), and the substrate can be Z-Gly-Gly-Arg-AMC. Forcathepsin B, the buffer can contain 50 mM sodium phosphate, 1 mM EDTA,and 10 mM 2-mercaptoethanol (pH 6.25), and the substrate can beZ-Arg-Arg-AMC.

The inhibition constants can then be calculated by using the followingequation, with an assumption that inhibition is fully competitive:

V _(i)=(V _(max) [S])/([S]+K _(m)(1+[I]/K _(i))

where V_(i) is the observed residual activity, [S] is the substrateconcentration used in the assay, V_(max) is the maximal velocity at aninhibitor concentration of zero, K_(i) is the inhibitor dissociationconstant, and [I] is the inhibitor concentration. Curves can then befitted by nonlinear regression analysis by using fixed values for thesubstrate concentration and the value of the Michaelis constant (K_(m)).Data analysis can be carried out by using Prism v 2.01 (GraphPad, SanDiego, Calif.).

The term “IC₅₀” indicates how much of a compound is needed to inhibit agiven biological process (or component of a process, e.g., an enzyme,cell, cell receptor, or microorganism) by one half (50%). The IC₅₀ of acompound can be determined by constructing a dose-response curve andexamining the effect of different concentrations of the compound onreversing the activity of the enzyme. From the dose-response curve, IC₅₀values can be calculated for a given compound by determining theconcentration needed to inhibit half of the maximum biological responseof the enzyme.

In general, the Kgp Ki value for compounds of the invention ranges fromabout 0.001 nM to about 500 nM. The Kgp Ki value for a compound of theinvention can range, for example, from about 1 nM to about 20 nM, orfrom about 20 nM to about 40 nM, or from about 40 nM to about 60 nM, orfrom about 60 nM to about 80 nM, or from about 80 nM to about 100 nM, orfrom about 100 nM to about 150 nM, or from about 150 nM to about 200 nM,or from about 200 nM to about 250 nM, or from about 250 nM to about 300nM, or from about 300 nM to about 350 nM, or from about 350 nM to about400 nM, or from about 400 nM to about 450 nM, or from about 450 nM toabout 500 nM. The Kgp Ki value for a compound of the invention can rangefrom about 0.001 nM to about 0.025 nM, or from about 0.025 nM to about0.050 nM, or from about 0.050 nM to about 0.075 nM, or from about 0.075nM to about 0.100 nM, or from about 0.100 nM to about 0.250 nM, or fromabout 0.250 nM to about 0.500 nM, or from about 0.500 nM to about 0.750nM, or from about 0.750 nM to about 1 nM.

In general, the Kgp IC₅₀ value for compounds of the invention rangesfrom about 0.001 nM to about 500 nM. The Kgp IC₅₀ value for a compoundof the invention can range, for example, from about 1 nM to about 20 nM,or from about 20 nM to about 40 nM, or from about 40 nM to about 60 nM,or from about 60 nM to about 80 nM, or from about 80 nM to about 100 nM,or from about 100 nM to about 150 nM, or from about 150 nM to about 200nM, or from about 200 nM to about 250 nM, or from about 250 nM to about300 nM, or from about 300 nM to about 350 nM, or from about 350 nM toabout 400 nM, or from about 400 nM to about 450 nM, or from about 450 nMto about 500 nM. The Kgp IC₅₀ value for a compound of the invention canrange from about 0.001 nM to about 0.025 nM, or from about 0.025 nM toabout 0.050 nM, or from about 0.050 nM to about 0.075 nM, or from about0.075 nM to about 0.100 nM, or from about 0.100 nM to about 0.250 nM, orfrom about 0.250 nM to about 0.500 nM, or from about 0.500 nM to about0.750 nM, or from about 0.750 nM to about 1 nM.

In some embodiments, a Kgp inhibitor according to the invention has aKgp Ki of 100 nM or less. In some embodiments, the Kgp inhibitor has aKgp Ki of 50 nM or less.

In some embodiments, a Kgp inhibitor according to the invention has aKgp IC₅₀ of 50 nM or less. In some embodiments, the Kgp inhibitor has aKgp IC₅₀ of 15 nM or less.

Compounds having Kgp Ki values of 15 nM or less can be particularlyuseful for systemic administration. For example, such compounds can haveKgp Ki values ranging from about 1 picomolar (pM) to about 15 nanomolar(nM), from about 10 pM to about 12 nM, from about 100 pM to about 11 nM,or from about 100 pM to about 10 nM. Such compounds can have Kgp Kivalues of less than 10 nanomolar (nM), less than 8 nM, less than 6 nM,or less than 4 nM.

Compounds having Kgp Ki values of 45 nM or less can be particularlyuseful for topical administration. For example, such compounds can haveKgp Ki values ranging from about 1 picomolar (pM) to about 40 nanomolar(nM), from about 10 pM to about 35 nM, from about 100 pM to about 30 nM,or from about 100 pM to about 25 nM.

In certain embodiments, Kgp inhibitors according to the invention areselective for Kgp. As used herein, a “selective” Kgp inhibitor is acompound that does not substantially affect the activity of proteasesother than Kgp, RgpA, and RgpB when administered at a therapeuticallyeffective dose for treating a disease or condition associated with P.gingivalis infection. Typically, a protease that is not substantiallyaffected by a particular compound exhibits at least 90% of its normalenzymatic activity in the presence of the compound under physiologicalconditions. Selective Kgp inhibitors include those compounds that do notaffect the activity of proteases other than Kgp when administered at atherapeutically effective dose for treating a brain disorder,periodontal disease, diabetes, a cardiovascular disease, arthritis,preterm birth, pneumonia, cancer, a kidney disease, a liver disease, aretinal disorder, or glaucoma associated with P. gingivalis infection.Preferably, selective Kgp inhibitors do not adversely affect thecoagulation cascade when administered at therapeutically effectivelevels.

In some embodiments, the invention provides a Kgp inhibitor having a KgpKi of less than 50 nM. In some such embodiments, the trypsin Ki isgreater than 60 nM. In the some embodiments, the Kgp inhibitor has a Kifor Kgp of less than 15 nM, and a (trypsin Ki)/(Kgp Ki) ratio of greaterthan 100.

In some embodiments, the invention provides compounds that are at least30 times more selective for Kgp than for trypsin or cathepsin B. Forsome such compounds, the Kgp Ki is 0.9 nM, and the trypsin Ki and/or thecathepsin B Ki are 30 nM or more. In some embodiments, the Kgp Ki is 0.9nM, and the trypsin Ki and/or the cathepsin B Ki are 115 μM or more. Forsome such compounds, the Kgp IC₅₀ is 50 nM or less and the trypsin IC₅₀trypsin is 100 nM or more. For some such compounds, the Kgp IC₅₀ is 15nM or less and the trypsin IC₅₀ trypsin is 1 μM or more.

IV. Methods of Making Compounds

Certain examples of compounds of Formula (I) can be prepared startingwith certain lysine derivatives D1 and D6, which are described below andare commercially available or can be prepared following publishedprocedures.

In D1, preferred R⁷ and R⁸ each can be removed by chemical conditionsthat do not remove the other. For example, R⁷=benzyl can be removed byhydrogen and a palladium-carbon catalyst, but R⁷ is not affected bytrifluoroacetic acid, whereas R⁸=t-butyl can be removed bytrifluoroacetic acid, but R⁸ is not affected by hydrogen and apalladium-carbon catalyst. Other appropriate, complimentary combinationsof R⁷ and R⁸ have been published. Similarly, in D6, complimentary,removable R⁸ and R⁹ are preferred, and several appropriate combinationshave been published.

Certain D5 can be prepared by a sequence of transformations D1 to D2 toD3 to D4 to D5. See, Scheme 1.

In most instances, the transformation of D1 to D2 will involve more thanone chemical reaction. Following published procedures, the followingchemical reactions can be applied to transform D1 to D2. D1 can beconverted to E1 by treatment with N-methyl-O-methylhydroxylaminehydrochloride, an organic base (for example Et₃N), a racemizationinhibitor (for example HOBt), and a dehydrating agent (for exampleEDAC), in an organic solvent (for example DMF). See, Scheme 2, step (a).E1 can be converted to D2.5 by treatment with a lithiated heterocycle(for example 2-lithiobenzothiazole, 2-lithiothiazole, or2-lithiopyridine), in an organic solvent (for example THF), to installthe corresponding R¹⁰ (2-benzothiazolyl, 2-thiazolyl, or 2-pyridyl).See, Scheme 2, step (b).

D1 can be converted to E2 by treatment with ammonium hydrochloride, anorganic base (for example Et₃N), a racemization inhibitor (for exampleHOBt), and a dehydrating agent (for example EDAC), in an organic solvent(for example DMF). See, Scheme 2, step (c). E2 can be converted to D2-6by treatment with an organic base (for example Et₃N), and a strongdehydrating agent (for example pyridine-sulfur trioxide complex), in anorganic solvent (for example CH₂C12). See, Scheme 2, step (d).

D1 can be converted to D2-8 by treatment with fluoroacetic anhydride,and Et₃N, and DMAP, in an organic solvent (for example DMF). See, Scheme2, step (e).

D1 can be converted to E3 by treatment with borane-dimethylsulfidecomplex, in an organic solvent (for example THF). See, Scheme 3, step(a). E3 can be converted to E7 by treatment with an organic base (forexample Et₃N), a strong dehydrating agent (for example oxalyl chloride),and dimethylsulfoxide, in an organic solvent (for example CH₂Cl₂). See,Scheme 3, step (b). E7 can be converted to D2-10 by treatment withtrimethyl diazo phosphonacetate and K₂CO₃, in an alcohol solvent (forexample methanol). See, Scheme 3, step (c).

D1 can be converted to E4 by treatment with an organic base (for exampleEt₃N), a chloroformate (for example EtO₂CCl), and diazomethane, in anorganic solvent (for example diethyl ether). See, Scheme 4, step (a). E4can be converted to E11 by treatment with HBr, and acetic acid, in anorganic solvent (for example THF). See, Scheme 4, step (b). E11 can beconverted to D2-9 by treatment with an alcohol HOR¹¹ (for example2,3,5,6-tetrafluorophenol), and KF, in an organic solvent (for exampleDMF), to install the corresponding —OR¹¹ (for example2,3,5,6-tetrafluorophenoxy). See, Scheme 4, step (c).

Furthermore, a wide variety of additional published procedures can beapplied to transform D1 to other D2, in which X is a thiol-reactivegroup that is not illustrated.

After transformation of D1 to D2 (e.g., to D2-5, D2-6, D2-8, D2-9, orD2-10), R⁷ can be removed by appropriate chemical conditions, generatingD3 after spontaneous decarboxylation. D3 or a salt of D3 (e.g., thehydrochloride salt of D3) can be used in further synthetic steps. D3 canbe treated with a carboxylic acid R³CO₂H, and a racemization inhibitor(for example HOBt), and a dehydrating agent (for example EDAC), in anorganic solvent (for example DMF), generating D4. Alternatively, D3 canbe treated with R³COX, wherein X is a leaving group (for examplechloride), and an organic base (for example Et₃N), in an organic solvent(for example CH₂C12), generating D4. Alternatively, D3 can be treatedwith an isocyanate in organic solvent (for example CH₂C12), generatingD4. A wide variety of applicable R³CO₂H, R³COX, and isocyanates arecommercially available, or can be prepared by published procedures. R⁸can be removed by an appropriate chemical conditions, generating D5after spontaneous decarboxylation.

Other D5 can be prepared by a sequence of transformations D6 to D7 to D8to D4 to D5. See, Scheme 5.

D6 can be treated with a carboxylic acid R³CO₂H, and a racemizationinhibitor (for example HOBt), and a dehydrating agent (for exampleEDAC), in an organic solvent (for example DMF), generating D7. See,Scheme 5, step (a). Alternatively, D6 can be treated with R³COX, whereinX is a leaving group (for example chloride), and an organic base (forexample Et₃N), in an organic solvent (for example CH₂C12), generatingD7. Alternatively, D3 as shown in Scheme 1 can be treated with anisocyanate in organic solvent (for example CH₂C12), generating D7. Awide variety of applicable R³CO₂H, R³COX, and isocyanates arecommercially available, or can be prepared by published procedures. R⁹can be removed by appropriate chemical conditions generating D8. See,Scheme 5, step (b).

D8 can be transformed to D4 by sequences of reactions similar to thosedescribed for transformation of D1 to D2. See, Scheme 5, step (c). Insome embodiments, D8 is transformed to D4-9 as shown in Scheme 6, steps(a)-(c). In some embodiments, —OR¹¹ in D4-9 is2,3,5,6-tetrafluorophenoxy.

Furthermore, a wide variety of additional published procedures can beapplied to transform D8 to other D4, in which X is a thiol-reactivegroup that is not illustrated. Following the alternative sequence to D4,R⁸ can be removed by appropriate chemical conditions, generating D5after spontaneous decarboxylation. See, Scheme 5, step (d).

Preparation of certain examples of Formula (I) will require initialpreparation of unnatural amino acids that feature side-chains that arenot present in any of the amino acids that occur in proteins. A widevariety of methods have been published for preparation of amino acidsthat feature unnatural side-chains, including the most useful andimportant methods F1-4:

Although not illustrated in F1-3, the amine and carboxylate groups aretypically protected before application of these methods, and theprotection is removed after construction of the unnatural side-chain. InF1, a natural side-chain (R) is modified to form an unnatural side-chain(R′). The natural amino acids serine, glutamic acid, and methioninewould be especially for preparation of certain examples of Formulas (I)and (II). In F2, a metalated glycine derivative is treated with analkylating agent (R′X) to install the unnatural side-chain (R′). In someinstances, the metalated glycine derivative is generated by treating aglycine derivative with a strongly basic metalating agent (for examplelithium diisopropylamide or potassium t-butoxide). In other instances,the starting glycine derivative is sufficiently acidic that a much lessbasic metalating agent (for example potassium carbonate) issatisfactory. In the latter instances the metalated glycine derivativemay exist as a dissociated ion pair, rather than as the covalentlybonded species illustrated in F2. In F3, a metalated alanine derivativeis treated with an alkylating agent (R′X) to install the unnaturalside-chain (R′). In most instances, the metalated alanine derivative isgenerated by treating a halogenated alanine derivative with a low valentmetal (for example zinc dust). In many instances, a soluble palladiumcatalyst is utilized to facilitate F3. In F4, an aldehyde (R′CHO) isreacted with a source of ammonia and a source of cyanide, to generate anamino-nitrile, which is subsequently hydrolyzed to generate anamino-acid featuring an unnatural sidechain (R′). Such methods can beused to prepared intermediates C1, C2, C3, and C4 set forth above.

After application of appropriate methods to prepare amino acids thatfeature unnatural sidechains, these amino acids can be appropriatelyprotected, as described for D1 and D6, and then appropriate methods canbe applied, as described for D2-4 and D7-9, to generate analogs of D5,wherein the lysine side-chain has been replaced with an unnaturalside-chain. Thus, suitable methods are available to provide thevariations of the R³, Z, and CH₂AC(B)(D)CH₂NH₂ that are specified forFormulas (I) and (II).

V. Compositions/Administration

In a related aspect, the invention provides a pharmaceutical compositioncomprising a compound of Formula I and a pharmaceutically acceptableexcipient.

The pharmaceutical compositions can be prepared by any of the methodswell known in the art of pharmacy and drug delivery. In general, methodsof preparing the compositions include the step of bringing the activeingredient into association with a carrier containing one or moreaccessory ingredients. The pharmaceutical compositions are typicallyprepared by uniformly and intimately bringing the active ingredient intoassociation with a liquid carrier or a finely divided solid carrier orboth, and then, if necessary, shaping the product into the desiredformulation. The compositions can be conveniently prepared and/orpackaged in unit dosage form.

Pharmaceutical compositions containing compounds of the invention can beformulated for oral use. Suitable compositions for oral administrationinclude, but are not limited to, tablets, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, syrups, elixirs, solutions, buccal patches, oral gels,chewing gums, chewable tablets, effervescent powders, and effervescenttablets. Compositions for oral administration can be formulatedaccording to any method known to those of skill in the art. Suchcompositions can contain one or more agents selected from sweeteningagents, flavoring agents, coloring agents, antioxidants, and preservingagents in order to provide pharmaceutically elegant and palatablepreparations.

Tablets generally contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients, including: inertdiluents, such as cellulose, silicon dioxide, aluminum oxide, calciumcarbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose,calcium phosphate, and sodium phosphate; granulating and disintegratingagents, such as corn starch and alginic acid; binding agents, such aspolyvinylpyrrolidone (PVP), cellulose, polyethylene glycol (PEG),starch, gelatin, and acacia; and lubricating agents such as magnesiumstearate, stearic acid, and talc. The tablets can be uncoated or coated,enterically or otherwise, by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate can beemployed. Tablets can also be coated with a semi-permeable membrane andoptional polymeric osmogents according to known techniques to formosmotic pump compositions for controlled release.

Compositions for oral administration can be formulated as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent (such as calcium carbonate, calcium phosphate, or kaolin), or assoft gelatin capsules wherein the active ingredient is mixed with wateror an oil medium (such as peanut oil, liquid paraffin, or olive oil).

Kgp inhibitors can also be administered topically as a solution,ointment, cream, gel, or suspension, as well as in mouth washes,eye-drops, and the like. Still further, transdermal delivery of Kgpinhibitors can be accomplished by means of iontophoretic patches and thelike.

Pharmaceutical compositions containing Kgp inhibitors can also be in theform of a sterile injectable aqueous or oleaginous solutions andsuspensions. Sterile injectable preparations can be formulated usingnon-toxic parenterally-acceptable vehicles including water, Ringer'ssolution, and isotonic sodium chloride solution, and acceptable solventssuch as 1,3-butane diol. In addition, sterile, fixed oils can be used asa solvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic monoglycerides, diglycerides, ortriglycerides.

Aqueous suspensions can contain one or more Kgp inhibitors in admixturewith excipients including, but not limited to: suspending agents such assodium carboxymethylcellulose, methylcellulose,oleagino-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin, polyoxyethylene stearate, and polyethylene sorbitanmonooleate; and preservatives such as ethyl, n-propyl, andp-hydroxybenzoate. Dispersible powders and granules (suitable forpreparation of an aqueous suspension by the addition of water) cancontain one or more Kbp inhibitors in admixture with a dispersing agent,wetting agent, suspending agent, or combinations thereof. Oilysuspensions can be formulated by suspending a Kgp inhibitor in avegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil),or in a mineral oil (e.g., liquid paraffin). Oily suspensions cancontain one or more thickening agents, for example beeswax, hardparaffin, or cetyl alcohol. These compositions can be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention can also be in the formof oil-in-water emulsions. The oily phase can be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, such as gum acacia or gum tragacanth;naturally-occurring phospholipids, such as soy lecithin; esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate; and condensation products of said partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate.

Additionally, the present invention encompasses various administrationmodes by which the compounds can be delivered to increasebioavailability or blood brain barrier penetration, including but notlimited to, intravenous, intranasal, intrathecal, subcutaneous,intracranial and oral. Time release technology can be used to increasebioavailability including formulations for sustained-release (SR),sustained-action (SA), extended-release (ER, XR, XL) timed-release (TR),controlled-release (CR), modified release (MR), continuous-release,osmotic release and slow release implants. These alternative routes ofadministration were not previously considered for these molecules, whichwere primarily contemplated to be formulated for topical gingivaldelivery and not systemically.

The use of hybrid molecules to promote active transport or nanoparticlescan be used in certain embodiments to increase blood brain barriertransport. For example liposomes, proteins, engineered peptide compoundsor antibodies that bind to the receptors that transport proteins acrossthe blood brain barrier including LPR-1 receptor, transferrin receptor,EGF-like growth factor or glutathione transporter can be used toincrease penetration into the brain. Physical techniques includingosmotic opening, ultrasound, lasers, sphenopalantine ganglionstimulation, direct intracranial, intrathecal, or intraventriculardelivery via a pump can be used.

Pharmaceutical compositions according to the invention can also includeone or more additional active agents useful in the treatment ofconditions associated with P. gingivalis infection. In certainembodiments, the invention provides a pharmaceutical compositioncomprising one or more Kgp inhibitors as described herein in combinationwith one or more additional active agents for treatment of Alzheimer'sdisease. Several therapeutics are in development and in clinical use fortreatment of Alzheimer's disease. Therapeutic strategies includelowering circulating levels of β-amyloid and tau (as described in moredetail below), stabilizing microtubules, removing atheroscleroticplaques, modulating autophagy, modulating neurotransmitter levels, andinhibiting GABA(A) α5 receptors. Such therapeutics can maintain and/orrestore cognitive function in subjects with Alzheimer's disease; slowthe decline of cognitive function; and promote neuroplasticity andrecovery of the brain.

Active agents that can be combined with Kgp inhibitors in pharmaceuticalcompositions include, but are not limited to, antibiotics (i.e.,bacteriocidal compounds and bacteriostatic compounds), cholinesteraseinhibitors, alpha-7 nicotinic receptor modulators, serotonin modulators,NMDA modulators, Aβ-targeted therapies, ApoE-targeted therapies,microglia-targeted therapies, blood/brain barrier-targeted therapies,tau-targeted therapies, complement-targeted therapies, andanti-inflammatories.

Any suitable antibiotic can be combined with one or more Kgp inhibitorsin the pharmaceutical compositions of the invention. In certainembodiments, the invention provides a pharmaceutical compositioncontaining one more Kgp inhibitors and an antibiotic having a P.gingivalis MIC₅₀ of less than 25 μg/ml. For example, the P. gingivalisMIC₅₀ of the antibiotic can be less than 20 μg/ml, less than 15 μg/ml,less than 10 μg/ml, less than 8 μg/ml, less than 6 μg/ml, or less than 5μg/ml. In some embodiments, the P. gingivalis MIC₅₀ of the antibiotic isless than 1 μg/ml. In some embodiments, the P. gingivalis MIC₅₀ of theantibiotic is less than 0.2 μg/ml.

Examples of bacteriocidal and bacteriostatic compounds include, but arenot limited to: quinolones (e.g., moxifloxacin, gemifloxacin,ciprofloxacin, oflaxacin, trovafloxacin, sitafloxacin, and the like),β-lactams (e.g., penicillins such as amoxicillin,amoxacilin-clavulanate, piperacillin-tazobactam, penicillin G, and thelike; and cephalosporins such as ceftriaxone and the like), macrolides(e.g., erythromycin, azithromycin, clarithromycin, and the like),carbapenems (e.g., doripenem, imipenem, meropinem, ertapenem, and thelike), thiazolides (e.g, tizoxanidine, nitazoxanidine, RM 4807, RM 4809,and the like), tetracyclines (e.g., tetracycline, minocycline,doxycycline, eravacycline, and the like), clindamycin, metronidazole,and satranidazole. Bacteriocidal and bacteriostatic compounds alsoinclude agents that inhibit or otherwise interfere with formation ofbiofilms by anaerobic, gram-negative bacteria; such agents includeoxantel, morantel, thiabendazole, and the like. Compositions of theinvention can contain one or more Kgp inhibitors with one or more (e.g.,two, three, four, five, six, or more) bacteriocidal/bacteriostaticcompounds. Compositions containing bacteriocidal/bacteriostaticcompounds can further contain a chlorhexidine (e.g., chlorhexidinedigluconate) alone or in combination with a zinc compound (e.g., zincacetate), can also be used in combination with the administeredantibiotics.

In some embodiments, a combination of a penicillin (e.g., amoxicillin)and metronidazole or a combination of penicillin (e.g., amoxicillin),metronidazole and a tetracycline is used. In some embodiments, theantibiotic is selected from minocycline, doxycycline, metronidazole,amoxicillin, clindamycin, augmentin, satranidazole, and combinationsthereof.

Examples of suitable cholinesterase inhibitors include, but are notlimited to, donepezil, donepezil/memantine, galantamine, rivastigmine,and tacrine, as well as pharmaceutically acceptable salts thereof.Examples of suitable serotonin modulators include, but are not limitedto, idalopirdine, RVT-101, citalopram, escitalopram, fluoxetine,fluvoxamine, paroxetine, and sertraline, as well as pharmaceuticallyacceptable salts thereof. Examples of suitable alpha-7 nicotinicreceptor modulators include, but are not limited to, alpha-7 agonistssuch as encenicline and APN1125. Suitable NMDA modulators include, butare not limited to, NMDA receptor antagonists such as memantine andderivatives thereof.

Pharmaceutical compositions of the invention can also contain activeagents that are directed to biomolecular targets associated withneurological diseases. Such targets include beta amyloid peptides (alsoreferred to as beta amyloid, abeta, or Aβ), apolipoprotein E (alsoreferred to as ApoE), and microtubule-associated tau (also referred toas tau proteins, or simply as tau).

Aβ-targeted therapies include inhibitors of Aβ production (such asbeta-secretase inhibitors, gamma-secretase inhibitors, alpha-secretaseactivators), inhibitors of Aβ aggregation, inhibitors of Aβoligomerization, and upregulators of Aβ clearance, among others (see,e.g., Jia, et al. BioMed Research International, 2014. Article ID837157, doi:10.1155/2014/837157). Examples of Aβ-targeted therapiesinclude but are not limited to, antibodies, pioglitazone, begacestat,atorvastatin, simvastatin, etazolate, and tramiprosate, as well aspharmaceutically acceptable salts thereof.

Examples of ApoE-targeted therapies include, but are not limited toretinoid X receptor agonists (see, Cramer, et al., Science 2012.335(6075): 1503-1506) and others described by Liu et al. (Nat RevNeurol. 2013. 9(2): 106-118). Tau-targeted therapies include, but arenot limited to, methylthioninium, leuco-methylthioninium, antibodies andthose described by Lee, et al. (Cold Spring Harb Perspect Med 2011;1:a006437).

Pharmaceutical compositions of the invention can also containcomplement-targeted therapies. Such therapies target components of thecomplement system involved in the innate immune response. Complementtargeted therapies include, but are not limited to, those described byRicklin and Lambris (Nat. Biotechnology 2007. 25(11): 1265-1275).

Examples of suitable anti-inflammatories include, but are not limitedto, NSAIDs such as apazone, diclofenac, ibuprofen, indomethacin,ketoprofen, nabumetone, naproxen, piroxicam, and sulindac, as well aspharmaceutically acceptable salts thereof.

VI. Methods of Treatment

As described above, infection with P. gingivalis and gingipain activityhave been linked to the development of periodontal disease, Alzheimer'sand other brain disorders, cardiovascular disease, diabetes, cancer,liver disease, kidney disease, preterm birth, arthritis, pneumonia andother disorders. See: Bostanci, et al. FEMS Microbiol Lett, 2012.333(1): 1-9; Ghizoni, et al. J Appl Oral Sci, 2012. 20(1): 104-12; Gatz,et al. Alzheimers Dement, 2006. 2(2): 110-7; Stein, et al. J Am DentAssoc, 2007. 138(10): 1314-22; quiz 1381-2; Noble, et al. J NeurolNeurosurg Psychiatry, 2009. 80(11): 1206-11; Sparks Stein, et al.Alzheimers Dement, 2012. 8(3): 196-203; Velsko, et al. PLoS ONE, 2014.9(5): e97811; Demmer, et al. J Dent Res, 2015. 94(9S): 201-S-11S;Atanasova and Yilmaz. Molecular Oral Microbiology, 2014. 29(2): 55-66;Yoneda, et al. BMC Gastroenterol, 2012. 12: 16. Kgp inhibitors cantherefore be used to diseases and conditions, such as brain disorders,caused by or otherwise affected by P. gingivalis.

Accordingly, another aspect of the invention provides a method oftreating a disease or condition associated with P. gingivalis infection.The method includes administering to a subject an effective amount of acompound according to Formula Ie:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Z is a thiol-reactive group or a masked thiol-reactive group;    -   A is selected from —CH₂— and —O—;    -   B and D are independently selected from hydrogen, halogen, C₁₋₄        haloalkyl, and C₁₋₄ haloalkoxy;    -   R¹ is selected from hydrogen and an amine protecting group;    -   R² is hydrogen; and    -   R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₁₋₈ alkyl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, -L-R⁵, and —OR⁶, wherein    -   L is selected from —O—, —NR—, C₁₋₄ alkylene, and 2- to        4-membered heteroalkylene, wherein R is selected from hydrogen        and C₁₋₈ alkyl,    -   R⁵ is selected from C₆₋₁₀ aryl, 5-to-12 membered heteroaryl,        C₃₋₈ cycloalkyl, and 5-to-12 membered saturated heterocyclyl,        and    -   —OR⁶ and the carbonyl to which it is bonded form an amine        protecting group,    -   and wherein R³ is optionally substituted with one or more        substituents selected from halo, —CN, —NO₂, —N₃, —OH, R^(a),        R^(b), —OR^(a), —OR^(b), —(CH₂)_(k)C(O)R^(c),        —NR^(d)(CH₂)_(U)C(O)R^(c), —O(CH₂)_(u)C(O)R^(c),        —(CH₂)_(k)CONR^(d)R^(d), —(CH₂)_(k)NR^(d)C(O)R^(c),        —NR^(d)(CH₂)_(u)CONR^(d)R^(d), —NR^(d)(CH₂)_(u)NR^(d)C(O)R^(c),        —O(CH₂)_(u)CONR^(d)R^(d), —O(CH₂)_(u)NR^(d)C(O)R^(c),        —(CH₂)_(k)S(O)₂NR^(d)R^(d), —(CH₂)_(k)NR^(d)S(O)₂R^(c),        —(CH₂)_(k)S(O)₂R^(c), —(CH₂)_(k)S(O)R^(c), —(CH₂)_(k)SR^(d),        —NR^(d)(CH₂)_(u)S(O)₂NR^(d)R^(d),        —NR^(d)(CH₂)_(u)NR^(d)S(O)₂R^(c), —NR^(d)(CH₂)_(u)S(O)₂R^(c),        —NR^(d)(CH₂)_(u)S(O)R^(c), —NR^(d)(CH₂)_(u)SR^(d),        —O(CH₂)_(u)S(O)₂NR^(d)R^(d), —O(CH₂)_(u)NR^(d)S(O)₂R^(c),        —O(CH₂)_(u)S(O)₂R^(c), —O(CH₂)_(u)S(O)R^(c), and        —O(CH₂)_(u)SR^(c), wherein:    -   each R^(a) is independently selected from C₁₋₄ alkyl and C₁₋₄        haloalkyl,    -   each R^(b) is independently selected from C₃₋₆ cycloalkyl, C₃₋₆        halocycloalkyl, C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(c) is independently selected from —OH, C₁₋₈ alkyl, C₁₋₈        haloalkyl, C₃₋₈ cycloalkyl, C₃₋₈ halocycloalkyl, C₆₋₁₀ aryl,        (C₆₋₁₀ aryl)-(C₁₋₈ alkyl), 5-to-12 membered heteroaryl, and        5-to-12 membered saturated heterocyclyl,    -   each R^(d) is independently selected from hydrogen and C₁₋₈        alkyl,    -   each subscript k is independently selected from 0, 1, 2, 3, 4,        5, and 6, and    -   each subscript u is independently selected from 1, 2, 3, 4, 5,        and 6; and    -   R⁴ is selected from hydrogen, halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy,        C₁₋₄ haloalkyl, and C₁₋₄ haloalkoxy.

In some embodiments, the method includes administering one or morecompounds from Table 1 to a subject.

TABLE 1 Compounds for use in the treatment of conditions associated withP. Gingivalis infection. Com- pound No. Compound Structure  1

 2

 3

 4

 5/6

 7

 8

 9

10

11

12

13

14/15

16/17

18/19

20/21

22

23

24

25/26

27/28

29

30

31

32

33

34/35

36/37

38

39

40

41

42

43

44

45

46

47

48

In some embodiments, the invention provides a method of treating adisease or condition associated with P. gingivalis infection asdescribed above, wherein the subject is a human or a canine.

In some embodiments, the invention provides a method of treating adisease or condition associated with P. gingivalis infection asdescribed above, wherein the compound of Formula Ie has a structureaccording to Formula If:

In some embodiments, the invention provides a method of treating adisease or condition associated with P. gingivalis infection asdescribed above, wherein R⁴ is hydrogen and B and D are independentlyselected from hydrogen, halogen, halomethyl, and halomethoxy.

In some embodiments, Z is selected from benzothiazol-2-yl-carbonyl;thiazol-2-yl-carbonyl; oxazol-2-yl-carbonyl; benzooxazol-2-yl-carbonyl;pyridin-2-yl-carbonyl; pyrimidin-4-yl-carbonyl; pyrimidin-2-yl-carbonyl;isoxazol-5-yl-carbonyl; isoxazol-3-yl-carbonyl;1,2,4-oxadiazol-3-yl-carbonyl; 1,2,4-oxadiazol-5-yl-carbonyl; cyano;ethynyl; fluoromethyl-carbonyl; acyloxymethyl-carbonyl;aryloxymethyl-carbonyl; alkylsulfonyl-vinyl; and arylsulfonyl-vinyl;each of which is optionally substituted with one or more substituentsselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkyl, C₁₋₄ haloalkoxy,halogen, and —N₃.

In some embodiments, Z is selected from benzothiazol-2-yl-carbonyl,halogen-substituted aryloxymethyl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl.

In some embodiments, the method includes administering a compound havinga structure according to Formula Ic:

or a pharmaceutically acceptable salt thereof, wherein R³ is selectedfrom C₆₋₁₀ aryl, 5-to-12 membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12membered saturated heterocyclyl, and -L-R⁵, wherein L is C₁₋₄ alkylene.

In some embodiments, the compound used in the method is a compound ofFormula Ic, or a pharmaceutically acceptable salt thereof, wherein R³ isselected from cyclohexyl, cyclopentyl, morpholino, phenyl, piperidinyl,pyridinyl, tetrahydrofuranyl, tetrahydropyranyl,1,2,3,4-tetrahydronaphthyl, and thiazolyl, each of which is optionallysubstituted with 1-3 members selected from methyl, methoxy,trifluoromethyl, acetyl, and —N₃.

In some embodiments, the compound used in the method is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the method includes administering a compound havinga structure according to Formula Id:

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein R³ is selected from C₆₋₁₀ aryl, 5-to-12 membered        heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturated        heterocyclyl, and -L-R⁵,    -   wherein L is C₁₋₄ alkylene.

In some embodiments, the compound used in the method is a compound ofFormula Id, or a pharmaceutically acceptable salt thereof, wherein R³ isselected from cyclohexyl, cyclopentyl, morpholino, phenyl, piperidinyl,pyridinyl, tetrahydrofuranyl, tetrahydropyranyl,1,2,3,4-tetrahydronaphthyl, and thiazolyl, each of which is optionallysubstituted with 1-3 members selected from methyl, methoxy,trifluoromethyl, acetyl, and —N₃.

In some embodiments, the compound used in the method is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the compound used in the method is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the compound used in the method of the invention isa compound of Formula I, wherein Z is selected frompyridin-2-yl-carbonyl and thiazol-2-yl-carbonyl, and R³ is selected fromC₆₋₁₀ aryl and C₃₋₈ cycloalkyl. In some such embodiments, the compoundused in the method is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the compound used in the method of the invention isa compound of Formula I, wherein R⁴ is selected from C₁₋₄ alkyl and C₁₋₄haloalkyl. In some embodiments, the compound used in the method is

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound used in the method is selected from:

and pharmaceutically acceptable salts thereof.

Curtis et al. have described a compound dubbed A71561 (see, Infectionand Immunity, 2002. 70(12): 6968-6975). A71561 is a mixture of twodiastereomers:2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(S)-lysinyl)-benzothiazoleand2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(S)-lysinyl)-benzothiazole.A71561 is not a selective inhibitor for Kgp, as its affinity to humanTrypsin (Ki=30 nM) is too close to the affinity for Kgp (Ki=0.9 nM).There is no suggestion by Curtis that A71561 or structurally similarcompounds may be used to treat brain disorders (e.g., Alzheimer'sdisease), diabetes, cardiovascular disease, arthritis or retinaldisorders.

The method of the invention specifically encompasses the use of2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-lysinyl)-benzothiazole,2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-lysinyl)-benzothiazole(compounds of Formula (III) and (IV), respectively, and mixturesthereof, and pharmaceutically acceptable salts thereof,

In some embodiments, a compound of Formula (III) is free orsubstantially free (contains less than 100000 parts per million) of2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(S)-lysinyl)-benzothiazole.In some embodiments, the compound of Formula (III) consists of2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(S)-lysinyl)-benzothiazole:

In some embodiments, a compound of Formula (III) would comprise (and insome embodiments, consist of) a mixture of the of2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(S)-lysinyl)-benzothiazoleand of2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(R)-lysinyl)-benzothiazolestereoisomers:

The2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(S)-lysinyl)-benzothiazoleand of2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(R)-lysinyl)-benzothiazolestereoisomers may, e.g., be in a ratio greater than 20:1. In someembodiments, the ratio would be between 20:1 and 2:1. In someembodiments, the ratio would be less than 2:1.

In some embodiments, a compound of Formula (IV) would comprise (and incertain preferred embodiments, consist of)2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(S)-lysinyl)-benzothiazole:

Thus, the invention encompasses use of a compound of Formula (IV) whichis free or substantially free (contains less than 100000 parts permillion) of2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(R)-lysinyl)-benzothiazole.

In some embodiments, a compound of Formula (IV) comprises a mixture of2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(S)-lysinyl)-benzothiazoleand2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(R)-lysinyl)-benzothiazole:

The2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(S)-lysinyl)-benzothiazoleand2-(N—[N-(3-phenylpropanoyl)-(S)-nipecotinyl]-(R)-lysinyl)-benzothiazolecan, for example, be in a ratio greater than 20:1. In some embodiments,the ratio is between 20:1 and 2:1. In some embodiments, the ratio isless than 2:1.

In some embodiments, the disease or condition is selected from a braindisorder, periodontal disease, diabetes, a cardiovascular disease,arthritis, preterm birth, pneumonia, cancer, a kidney disease, a liverdisease, a retinal disorder, and glaucoma.

In some embodiments, the disease or condition is a brain disorder.

In some embodiments, the brain disorder is selected from Alzheimer'sdisease, Down's syndrome, epilepsy, autism, Parkinson's disease,essential tremor, fronto-temporal dementia, progressive supranuclearpalsy, amyotrophic lateral sclerosis, Huntington's disease, multiplesclerosis, mild cognitive impairment, age associated memory impairment,chronic traumatic encephalopathy, stroke, Lewy Body disease, multiplesystem atrophy, schizophrenia, and depression.

In some embodiments, the brain disorder is Alzheimer's disease.

In some embodiments, the method further includes administering to thesubject one or more active agents selected from a cholinesteraseinhibitor, a serotonin modulator, an NMDA modulator, an Aβ targetedtherapy, an ApoE targeted therapy, a microglia targeted therapy, a bloodbrain barrier targeted therapy, a tau targeted therapy, a complementtargeted therapy, and an anti-inflammatory.

In some embodiments, the disease or condition is periodontal disease. Insome embodiments, the disease or condition is a liver disease. In someembodiments, the liver disease is non-alcoholic steatohepatitis. In someembodiments, the disease or condition is a retinal disorder. In someembodiments, the retinal disorder is age-related macular degeneration.

In some embodiments, the disease or condition is cancer. In someembodiments, the cancer is breast cancer, oral cancer, pancreaticcancer, or glioblastoma multiforme.

In certain embodiments, compounds of the invention inhibit active Kgp inthe brain of a mammal, e.g., a human or an animal (e.g., a dog), and arecytoprotective or neuroprotective. By “neuroprotective,” it is meantthat the compounds prevent aberrant changes to neurons or death ofneurons. Compounds of the invention are therefore useful, e.g., intreatment of a brain disorder (e.g., a neurodegenerative disease (e.g.,Alzheimer's disease, Down's syndrome, epilepsy, autism, Parkinson'sdisease, essential tremor, fronto-temporal dementia, progressivesupranuclear palsy, amyotrophic lateral sclerosis, Huntington's disease,multiple sclerosis, mild cognitive impairment, age associated memoryimpairment, chronic traumatic encephalopathy, stroke, Lewy Body disease,multiple system atrophy, schizophrenia and depression, etc.), diabetes,cardiovascular disease, arthritis, retinal disorders (e.g., age relatedmacular degeneration) and glaucoma.

As evidenced by FIG. 1,2-(N—[N-(3-phenylpropanoyl)-(R)-nipecotinyl]-(R)-lysinyl)-benzothiazoleis effective at preventing cell death. As evidenced by FIG. 2, compound1 and compound 2 are effective at preventing cell death. In addition,modifications to these 1-(3-phenylpropionyl)piperidine-3-carboxylicacid-[4-amino-1-(benzothiazole-2-carbonyl butyl] amides can improve theproperties (e.g., neuroprotective properties) of the compounds of theinvention and/or make compounds of the invention more suitable forsystemic administration. Data from in vitro assays demonstrates thatthis type of compounds can be used to protect cells from gingipaininduced cell death (FIG. 2).

Kgp inhibitors as described herein can be administered at any suitabledose in the methods of the invention. In general, a Kgp inhibitor isadministered at a dose ranging from about 0.1 milligrams to about 1000milligrams per kilogram of a subject's body weight (i.e., about 0.1-1000mg/kg). The dose of Kgp inhibitor can be, for example, about 0.1-1000mg/kg, or about 1-500 mg/kg, or about 25-250 mg/kg, or about 50-100mg/kg. The dose of Kgp inhibitor can be about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950 or 1000 mg/kg. The dosages can be varied depending upon therequirements of the patient, the severity of the disorder being treated,and the particular formulation being administered. The dose administeredto a patient should be sufficient to result in a beneficial therapeuticresponse in the patient. The size of the dose will also be determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of the drug in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the typical practitioner. The total dosage can be dividedand administered in portions over a period of time suitable to treat tothe seizure disorder.

Kgp inhibitors can be administered for periods of time which will varydepending upon the nature of the particular disorder, its severity, andthe overall condition of the subject to whom the Kgp inhibitor isadministered. Administration can be conducted, for example, hourly,every 2 hours, three hours, four hours, six hours, eight hours, or twicedaily including every 12 hours, or any intervening interval thereof.Administration can be conducted once daily, or once every 36 hours or 48hours, or once every month or several months. Following treatment, asubject can be monitored for changes in his or her condition and foralleviation of the symptoms of the disorder. The dosage of the Kgpinhibitor can either be increased in the event the subject does notrespond significantly to a particular dosage level, or the dose can bedecreased if an alleviation of the symptoms of the disorder is observed,or if the disorder has been remedied, or if unacceptable side effectsare seen with a particular dosage.

A therapeutically effective amount of a Kgp inhibitor can beadministered to the subject in a treatment regimen comprising intervalsof at least 1 hour, or 6 hours, or 12 hours, or 24 hours, or 36 hours,or 48 hours between dosages. Administration can be conducted atintervals of at least 72, 96, 120, 144, 168, 192, 216, or 240 hours(i.e., 3, 4, 5, 6, 7, 8, 9, or 10 days). In certain embodiments,administration of one or more Kgp inhibitors is conducted in a chronicfashion over periods ranging from several months to several years.Accordingly, some embodiments of the invention provide a method oftreating a disease or condition associated with P. gingivalis infectionas described above, wherein the compound is administered to the subjectfor at least one year. In some embodiments, the compound is administeredto the subject for at least 10 years. In some embodiments, the compoundis administered to the subject for at least 60 years.

Administration of Kgp inhibitors according to the methods of theinvention typically results in the reduction of circulating levels ofactive Kgp in a subject and/or the reduction of active Kgp in the brain.In certain embodiments, administration of a Kgp inhibitor according tothe methods of the invention results in at least a 20% reduction ofcirculating levels of active Kgp and/or at least a 20% reduction ofactive Kgp in the brain. For example, the circulating levels of Kgpand/or the levels of Kgp in the brain are preferably reduced by fromabout 25% to about 95%, or from about 35% to about 95%, or from about40% to about 85%, or from about 40% to about 80% as compared to thecorresponding levels of Kgp 24 hours prior to the first administrationof the Kgp inhibitor.

Kgp inhibitors can be administered alone or in combination with one ormore additional therapeutically active agents, as described above. Theone or more additional therapeutically effective agents include, e.g.:(i) a pharmaceutically acceptable agent which inhibits ArginineGingipain A (RgpA) and/or Arginine Gingipain B (RgpB) production,translocation of RgpB and/or Kgp into systemic circulation or brainand/or pathological (e.g., neurotoxic effects) of RgpA, RgpB and or Kgpin a mammal; (ii) an antibacterial agent which is bacteriostatic orbacteriocidal with respect to P. gingivalis; (iii) one or moreantibodies which bind to RgpA, RgpB and/or Kgp (e.g., 18E6, which bindsto the first half of the immunoglobulin domain of RgpB; Kgp-specificmonoclonal antibody, 7B9, which recognizes an epitope within the Kgpcatalytic domain; the RgpA antibody 61Bg 1.3, humanized versions of anyof the foregoing, etc.); (iv) epitopes of antibodies which bind to RgpA,RgpB and/or Kgp or other proteins expressed by P. gingivalis; and (v)combinations of any of the foregoing.

The additional therapeutically active agents also include AP peptideslevel reducers, pathogenic level tau reducers, microtubule stabilizers,agents capable or removing atherosclerotic plaques, agents that lowercirculating levels of β-amyloid and tau, modulators of autophagy,neurotransmitter level regulators, GABA(A) α5 receptors inhibitors, andadditional agents that help maintain and/or restore cognitive functionand functional deficits of Alzheimer's disease, and/or slow down declinein cognitive functions and functional deficits in Alzheimer's disease.

Pharmaceutical compositions of the invention can contain one or more Kgpinhibitors as described herein in combination with ritonavir (RTV),which can increase bioavailability and increase blood brain barrierpenetration. For example, ritonavir is commonly combined with oralpeptidic HIV protease inhibitors to increase plasma levels by inhibitingthe P450 3A4 enzyme and thus decreasing first-pass metabolism (see,Walmsley, et al., N Engl J Med, 2002. 346(26): 2039-46). In addition,RTV binds to P-glycoprotein, a transmembrane efflux pump that is foundin many tissues, including the blood brain barrier, allowingco-administered compounds better access to the brain (see, Marzolini, etal., Mol Pharm, 2013. 10(6): 2340-9). Therefore, a combination of RTVand Kgp inhibitors can be used to increase plasma concentrations andbrain levels of the gingipain inhibitors. It is shown herein that oraladministration of RTV 15 minutes prior to the Kgp inhibitor, Kyt-36increases the half life (FIG. 9) therefore it is expected that RTV willalso increase the half life of other Kgp inhibitors.

In some embodiments, compounds of the invention can be administered withnatural gingipain inhibitors including melabaricone C, isolated fromnutmeg or polyphenolic compounds derived from plants, such as cranberry,green tea, apple, and hops can be administered in conjunction fortreatment or prevention of brain disorders. Naturally and unnaturallyoccurring antimicrobial peptides including: κ-casein peptide (109-137)34, histatin 5, and CL(14-25), CL(K25A) and CL(R24A, K25A), can also beadministered in conjunction with the Kgp inhibitors of the invention.(see, e.g., Taniguchi et al., Biopolymers, 2014. 102(5): 379-89).

Kgp inhibitors as described herein can be administered with antibodiestargeting gingipains or other P. gingivalis proteins. Antibodies mayrely on damage to the blood brain barrier for access to the brain orperipheral interference with gingpains and P. gingivalis propagation.Antibodies can also help to stimulate the efficacy of the immune systemin clearing the bacteria. New or existing antibodies to RgpA, RgpB, orKgp can be utilized including 18E6 and 7B9. An RgpA antibody 61BG 1.3has previously demonstrated efficacy topically in prevention ofrecolonization by P. gingivalis after periodontal treatment. See, Boothet al., Infect Immun, 1996. 64(2): 422-7. Antibodies would preferably behumanized for use in humans. Methods known to those in the field fordelivery of biologics to improve half-life and brain penetration can beused including, but not limited to, intravenous delivery, subcutaneousdelivery, intranasal delivery, intrathecal delivery, vector transport,and direct brain delivery.

The methods of the invention also encompass administration of Kgpinhibitors as described herein with one or more of the followingadditional therapeutically active agents or pharmaceutically acceptablesalts thereof: an arginine derivative; histatin 5; baculovirus p35; asingle point mutant of cowpox viral cytokine-response modifier (CrmA(Asp>Lys)); phenylalanyl-ureido-citrullinyl-valyl-cycloarginal(FA-70C1); (acycloxy)methyl ketone (Cbz-Phe-Lys-CH₂OCO-2,4,6-Me3Ph);peptidyl chloro-methyl ketones (e.g., chloromethyl ketone derivaties ofarginine, chloromethyl ketone derivatives of lysine, and the like);fluoro-methyl ketones; bromo-methyl ketones; ketopeptides;1-(3-phenylpropionyl)piperidine-3(R,S)-carboxylic acid[4-amino-1(S)-(benzothiazole-2-carbonyl)butyl]amide (A71561); azapeptidefumaramide; aza-peptide Michael acceptors; benzamidine compounds;acyclomethylketone; activated factor X inhibitors (e.g., DX-9065a);cranberry nondialysable fraction; cranberry polyphenol fraction;pancreatic trypsin inhibitor; Cbz-Phe-Lys-CH₂O—CO—2,4,6-Me3-Ph; E-64;chlorhexidine; zinc (e.g., zinc acetate); or a combination of two, threeor more of any of foregoing.

In some of these embodiments, Zn can enhance potency and selectivity ofthe compounds (e.g., chlorhexidine, benzamidine, etc.) used in themethods of the invention. Benzamidine compounds include, e.g., thefollowing compounds and derivatives thereof:

A lysine gingipain inhibitor of the invention can be administered in thesame composition as an additional therapeutically active agent.Alternatively, the additional therapeutically active agent can beadministered separately before, concurrently with, or afteradministration of the lysine gingipain inhibitor.

Similar to humans, P. gingivalis infection and periodontal disease isone of the most common infectious diseases affecting adult dogs andcats. Using adult beagle dogs, researchers demonstrated the existence ofRgp in plaque samples taken from beagle dogs given a specific soft dietto increase plaque formation on tooth surfaces. (See, e.g.: Davis andHead, Front Pharmacol, 2014. 5: 47; Reichart, et al., Journal ofPeriodontal Research, 1984. 19(1): 67-75; Kataoka, S., et al., FASEB J,2014 28(8): 3564-78.) Dogs and cats with P. gingivalis infection andgingipains in their brain and circulatory system may experienceperiodontal disease, mild cognitive impairment, age associated memoryimpairments, damage or generalized accelerated aging due to gingipaininduced cell death, which could be treated or prevented withcompositions of Formula I, II, III, IV or V.

VII. Methods of Detecting P. gingivalis and Diagnosing ConditionsAssociated with P. gingivalis Infection

The present invention also provides for a diagnostic test for gingipainsor P. gingivalis in the brain or patient samples in order to diagnose orpredict brain disorders, or to determine who would be the bestcandidates for treatment with compounds described herein. Prior art hasnoted changes in serum profiles based on infection with P. gingivalis. Anovel assay to diagnose or predict risk of development of braindisorders is to conduct an ELISA on saliva, cerebral spinal fluid orblood, for example, to detect one or both gingipains. Saliva, blood andCSF levels of gingipain or other P. ginigivalis markers would beexpected to be higher in at risk patients and patients who are goodcandidates for treatment. Development of an ELISA is a fairly simpleprocess known to those skilled in the art utilizing one antibody againstthe target to capture the target and a second labeled antibody against adifferent epitope on the target to obtain a quantitative readout.Commercially available or newly generated antibodies could be used forthis purpose. Immobilized or labeled compounds described herein (forexample with biotin or HRP) could be utilized to substitute for one orboth antibodies. Click chemistry compounds such as those depicted inFIG. 10 could be utilized for this purpose. The diagnostic could includedetection of one or more gingipains. Biotinylation of the detectionantibody can be used to increase sensitivity.

Alternatively, instead of detecting the presence or absence of thegingipains, an assay for their activity in saliva, CSF or blood could beused. This would provide the benefit of providing a readout on the mostbiologically relevant factor (e.g., activity) in the presence or absenceof treatment, for example. Methods for developing enzyme assays areknown to those skilled in the art. A salivary test known as the BANATest is commercially available for dental applications to test forproteases from P gingivalis and 2 other oral bacteria. The BANA test isa small plastic card to which is attached two separate reagent matrices,seen as strips on the card. The lower white reagent matrix isimpregnated with N-benzoyl-DL-arginine-B-naphthylamide (BANA).Subgingival plaque samples are applied to the lower matrix, and thendistilled water is applied to the upper matrix. Then the lower matrix isfolded back to make contact with the upper matrix. The upper buffreagent matrix contains a chromogenic diazo reagent which reacts withone of the hydrolytic products of the enzyme reaction forming a bluecolor. The reaction occurs when the plastic strip is inserted into anincubator set at 35 degrees C. for 5 minutes. The BANA substrate detectsat least 3 different oral bacteria however and is not specific to P.gingivalis. The BANA test could be used to identify people at risk forbrain disorders or eligible for treatment. Alternatively, the BANAsubstrate can be substituted in similar formats or in a liquid assaywith an RgpA, RgpB and/or KGP specific substrate.

Reagents that bind to active gingipains, including but not limited tothose described in this application, can be used to precipitate onlyactive gingipains followed by detection with a monoclonal for example.Alternatively, an antibody or other high affinity binding agent could beused to precipitate the gingipain from the CSF followed by a proteaseassay with a labeled substrate, which allows for increased fluorescenceor colorometric readout as the labeled substrate is digested.

The present invention also provides for a diagnostic based on imaging P.gingivalis or its gingpains in the human brain. Any agent that binds togingipains, including but not limited to compounds of the presentinvention and other compounds described elsewhere, can be labeled withF18 or other radiographic markers and visualized using PET or SPECTscanning. A positive signal would indicate treatment with compoundsdescribed herein. In a preferred and non limiting embodiment, compound45 as described herein is modified via “click chemistry” to install aradiolabel that can be imaged with PET or SPECT (FIG. 10).

VIII. Examples Example 1

SH-SYSY neuroblastoma cells were cultured and differentiated in thepresence of 5 uM retinoic acid based on established methods [Saberi S.,et al. Cell Mol Neurobiol 2013. 33: 747-751]. Differentiation intoneuronal cells was verified by observation of neurite outgrowth. Thedifferentiated cells were exposed to 100 nM Kgp and/or RgpB for 24hours, in the presence or absence of Kgp inhibitors. Results wererecorded using a digital microscope camera (FIG. 1C, FIG. 2C).Gingipains are toxic to cells while COR compounds prevent thecytotoxicity.

Example 2

Adult male mice (CD-1, 25 g approximately) n=6 per group wereanaesthetized and injected unilaterally intrahippocampally usingstandard stereotaxic techniques. Gingipains RgpB and Kgp, purified fromP. gingivalis were diluted prior to injection to 10 μg/ml. Seven dayspost-surgery the animals were anaesthetized, perfused and humanelykilled and brains removed and sectioned for histological analysis.Fluoro-Jade staining was then be performed on sections of hippocampus toassess for neurodegeneration (Schmued L C and Hopkins K J, 2000).Fluoro-Jade staining identifies cell bodies, dendrites, axons and axonsterminals of degenerating neurons but does not stain healthy neurons,myelin, or vascular elements.

Brain sections were examined with an epifluorescence microscope (NikonMicrophot FXA) using a filter system suitable for visualizingfluorescein or fluorescein isothiocyanate (FITC). Images were acquiredwith a Leica DC Camera and an Image Analysis software (Leica IM50).Fluoro-Jade C—positive degenerating neurons appeared bright yellow-greenagainst a dark background and were clearly identified in the animalgroups treated with Gingipains. No Fluoro-Jade C—positive cells wereobserved in vehicle-treated group (FIG. 4).

Example 3

Female Balb/c mice were obtained from Harlan Laboratories (USA) andallowed to acclimate. 8 week old mice were challenged orally with 10⁹CFU W83 P. gingivalis in 2% Na-CMC, 2 times per week for 6 weeks.Control mice received mock challenge with 2% Na-CMC only. 6 weeks afterinitial infection, mice were sacrificed, perfused and brains dissected.Brains were embedded and sectioned. 18E6 immunohistochemistry for RgpBshowed brain infiltration in 3/6 mice. De Olmos silver stain forneurodegeneration showed staining in 2 of the 3 mice with infiltration(FIG. 5).

Example 4

Female Balb/cJ mice were obtained from Taconic and allowed to acclimate.8 week old mice were challenged orally with 10⁹ CFU W83 P. gingivalisevery 3rd day for 4 administrations.

Novel object recognition test for cognitive function was initiated 6weeks after the initial infection. Mice were familiarized with the testcage for 2 min the day prior to object familiarization. On the day offamiliarization, mice were presented with two wooden blue rectangles for5 minutes. 24 h later mice were presented with one blue rectangle (rightside) and one pink heart (left side, both objects made of wood) for theduration of 3 min. The time during which the mouse directed its nosewithin 2 cm of an object was recorded. Mock infected mice on averagespent more time exploring the novel object compared to infected mice whoon average spent equal time on both objects indicating cognitivedysfunction (FIG. 6).

Example 5

Female Balb/cJ mice were obtained from Taconic and allowed to acclimate.43-44 week old mice were challenged orally with 10⁹ CFU W83 P.gingivalis or a P. gingivalis strain lacking expression of Kgp everyother day for 42 days. Compound 2 or vehicle (2% DMSO) was givensubcutaneously three times per day on days 21-42. On day 42 mice weresacrificed and the brain removed and frozen. A sample includinghippocampal tissue was lysed in RIPA buffer with protease inhibitors andanalyzed for Abeta42 expression with the Amyloid beta 42 ELISA Kit,Mouse (Life Technologies). Infected mice showed an increase in abeta42that was not apparently in Kgp knock out mice or mice treated withcompound 2 (FIG. 7).

Example 6

Aged dogs were assessed for cognitive impairment as described by Araujoet al. [J Alzheimers Dis, 2011. 26(1): 143-55]. Brains were embedded andsliced and assessed for gingipain infiltration as described in Example 1with 7B9 antibody for Kgp (FIG. 8).

Example 7. Preparation ofbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate

(2S)-6-(benzyloxycarbonylamino)-2-(tert-butoxycarbonylamino)hexanoicAcid (ii)

To a solution of (2S)-6-amino-2-(tert-butoxycarbonylamino)hexanoic acid(100.00 g, 406.01 mmol, 1.00 eq), NaHCO₃ (102.33 g, 1.22 mol, 3.00 eq)in THF (1000 mL) and H₂O (1000 mL) was added CbzOSu (101.19 g, 406.01mmol, 1.00 eq) at 0° C. Then the mixture was stirred at 30° C. for 16hr. The mixture was adjusted to pH=5-6 with 1N HCl, extracted with EA(600 mL×3), dried over Na₂SO₄, concentrated to give(2S)-6-(benzyloxycarbonylamino)-2-(tert-butoxycarbonyl-amino)hexanoicacid (142.00 g, 373.26 mmol, 91.9% yield) as a yellow oil.

Tert-butyl-N-[(1S)-5-(benzyloxycarbonylamino)-1-[methoxy(methyl)-carbamoyl]pentyl]carbamate(iii)

To a solution of(2S)-6-(benzyloxycarbonylamino)-2-(tert-butoxycarbonylamino) hexanoicacid (142.00 g, 373.26 mmol, 1.00 eq), N-methoxymethanamine (54.61 g,559.89 mmol, 1.50 eq), EDCI (93.02 g, 485.24 mmol, 1.30 eq), HOBt (65.57g, 485.24 mmol, 1.30 eq) in DCM (2 L) was added TEA (113.31 g, 1.12 mol,3.00 eq) dropwise at 0° C. over 0.5 hr. Then the mixture was stirred at30° C. for 16 hr. TLC indicated the reaction completed. Then the mixturewas diluted with water (1000 mL) and separated. The organic layer waswashed with HCl (aq, 1M, 500 mL) and saturated NaHCO₃ (aq, 500 mL) insequence, dried over Na₂SO₄ and concentrated to give the crude, whichwas purified by column chromatography on silica gel (PE:EA=5: 1 toPE:EA=1: 1) to givetert-butyl-N-[(1S)-5-(benzyloxycarbonyl-amino)-1-[methoxy(methyl)carbamoyl]pentyl]carbamate(110.00 g, 259.74 mmol, 69.6% yield) as a light yellow oil.

Tert-butyl-N-[(1R)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonyl-amino)pentyl]-carbamate(iv)

To a mixture of 1,3-benzothiazole (9.58 g, 70.83 mmol, 3.00 eq) in THF(50 mL) was added n-BuLi (2.5 M in THF, 28.3 mL) dropwise at −65° C.under N₂. The mixture was stirred at −65° C. under N₂ for 1 hr. Thentert-butyl-N-[(1R)-5-(benzyloxycarbonylamino)-1-[methoxy(methyl)carbamoyl]pentyl]carbamate(10.00 g, 23.61 mmol, 1.00 eq) in THF (50 mL) was added dropwise at −65°C. under N₂ and the reaction mixture was stirred at −65° C. under N₂ foranother 3 hr. TLC indicated the reaction completed. The mixture wasquenched by saturated NH₄Cl (aq, 10 mL), then the mixture was extractedwith EA (100 mL×3), dried over Na₂SO₄ and concentrated to give thecrude, which was purified by column chromatography on silica gel(PE:EA=5: 1 to PE:EA=3: 1) to givetert-butyl-N-[(1R)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]-carbamate(6.20 g, 12.46 mmol, 52.8% yield) as a light yellow oil.

Benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(Core 1)

To a solution oftert-butyl-N-[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamate (10.84 g, 21.78 mmol, 1.00 eq) in EA (140 mL) was addedHO/EA (4 M, 12 mL). Then the reaction was stirred at 30° C. for 15 hr.The reaction mixture was filtered and the filter cake was washed with EA(80 mL) and dried in vacuo to affordbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (7.52 g, 17.33 mmol, 79.6% yield) as a yellow solid.

Example 8. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]benzamide (1)

Benzyl-N-[(5S)-5-benzamido-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(1a)

To a mixture ofbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]-carbamate;hydrochloride (400 mg, 921.77 μmol, 1.00 eq), TEA (280 mg, 2.77 mmol,3.00 eq) in DCM (10 mL) was added benzoyl chloride (156 mg, 1.11 mmol,1.20 eq) dropwise at 0° C. under N₂. The mixture was stirred at 0° C.for another 0.5 hr. TLC (PE:EA=5:1) indicated the reaction completed.The mixture was washed with water (5 mL×3), dried over Na₂SO₄,concentrated to givebenzyl-N-[(5S)-5-benzamido-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(300 mg, 598.09 μmol, 64.9% yield) as a light yellow solid.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]benzamide (1)

A mixture ofbenzyl-N-[(5S)-5-benzamido-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(250 mg, 498.41 μmol, 1.00 eq) in AcOH (1 mL) was added HBr/AcOH (5.5 M,1 mL). The mixture was stirred at 30° C. for 0.5 h. LC-MS indicated thereaction completed. Water (20 mL) was added and extracted with MTBE (5mL×2). Then the aqueous layer was lyophilized to give the crude, whichwas purified by prep-HPLC (CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]benzamidetrifluoroacetate (20 mg, 41.54 μmol, 8.33% yield) as a light yellowsolid. MS m/z=368.1 (MH⁺).

Example 9. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]cyclohexanecarboxamide(2)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(cyclohexanecarbonylamino)-6-oxo-hexyl]carbamate(2a)

To a mixture ofbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate;hydrochloride (400 mg, 921.77 μmol, 1.00 eq), cyclohexanecarboxylic acid(142 mg, 1.11 mmol, 1.20 eq), EDCI (230 mg, 1.20 mmol, 1.30 eq), HOBt(162 mg, 1.20 mmol, 1.30 eq) in DCM (10 mL) was added TEA (280 mg, 2.77mmol, 3.00 eq) dropwised at 0° C. The mixture was stirred at 30° C. for16 h. LC-MS indicated the reaction completed. Water (10 mL) was added,then extracted with DCM (10 mL×2), washed with water (10 mL), thensaturated NaHCO₃ (aq, 10 mL x 2), dried over Na₂SO₄, concentrated togive a crude, which was purified by flash chromatography to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(cyclohexanecarbonylamino)-6-oxo-hexyl]carbamate(2a; 210 mg, crude) as a yellow solid.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]cyclohexane-carboxamide(2)

To a mixture ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(cyclohexanecarbonylamino)-6-oxo-hexyl]carbamate(200 mg, 393.98 μmol, 1.00 eq) in AcOH (1 mL) was added HBr/AcOH (5.5 M,1 mL). The mixture was stirred at 30° C. for 0.5 hr. LC-MS indicated thereaction completed. Water (20 mL) was added and extracted with MTBE (5mL×2). Then the aqueous layer was lyophilized to give a crude, which waspurified by prep-HPLC to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]cyclohexanecarboxamidetrifluoroacetate (2; 10 mg, 20.51 μmol, 5.21% yield) as a light yellowsolid. MS m/z=374 (MH⁺). ¹H NMR (CD₃OD, 400 MHz) d 8.20 (dd, J=7.6,J=0.4, 1H), 8.14 (dd, J=7.6, J=1.2, 1H), 7.66-7.61 (m, 2H), 5.64 (dd,J=4.0, J=10.0, 1H), 3.02-2.93 (m, 2H), 2.38-2.35 (m, 1H), 2.18-2.10 (m,1H), 1.84-1.55 (m, 9H), 1.48-1.17 (m, 6H).

Example 10. Preparation of(3S)—N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoyl)piperidine-3-carboxamide(3)

Tert-butyl-(3S)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(3c)

A mixture of (3S)-1-tert-butoxycarbonylpiperidine-3-carboxylic acid (190mg, 829.59 μmol, 1.20 eq), HATU (315 mg, 829.59 μmol, 1.20 eq), DIPEA(268 mg, 2.07 mmol, 3.00 eq) in DMF (5 mL) was stirred at 0° C. for 1 h.Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the mixturewas stirred at 30° C. for 16 h. TLC (PE:EA=3:1) indicated the reactioncompleted. EA (20 mL) was added and washed with water (10 mL×3), driedover Na₂SO₄, concentrated to givetert-butyl-(3S)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(250 mg, 410.68 μmol, 59.4% yield, 55% de) as a yellow solid.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-piperidine-3-carbonyl]amino]hexyl]carbamate(3b)

To a mixture oftert-butyl-(3S)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(250 mg, 410.68 μmol, 1.00 eq) in EA (2 mL) was added HCl/EtOAc (4M, 2mL). The mixture was stirred at 30° C. for 1.5 h. TLC (PE:EA=3:1)indicated the reaction completed. The reaction mixture was filtered togivebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-piperidine-3-carbonyl]amino]hexyl]carbamate(3b; 200 mg, crude, 67% de) as a white solid.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-1-(3-phenylpropanoyl)-piperidine-3-carbonyl]amino]hexyl]carbamate(3a)

To a mixture ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-piperidine-3-carbonyl]amino]hexyl]carbamate(200 mg, 393.21 μmol, 1.00 eq), TEA (119 mg, 1.18 mmol, 3.00 eq) in DCM(5 mL) was added 3-phenylpropanoyl chloride (80 mg, 471.85 μmol, 1.20eq) at 0° C. under N₂. The mixture was stirred at 0° C. for another 0.5h. LC-MS indicated the reaction completed. DCM (10 mL) was added andwashed with water (5 mL×3), dried over Na₂SO₄, concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-1-(3-phenylpropanoyl)piperidine-3-carbonyl]amino]hexyl]carbamate(3a; 220 mg, crude) as a yellow solid.

(3S)—N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenyl-propanoyl)piperidine-3-carboxamide;2,2,2-trifluoroacetic acid (3)

To a mixture ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-1-(3-phenylpropanoyl)piperidine-3-carbonyl]amino]hexyl]carbamate (220 mg, 343.33 μmol, 1.00eq) in AcOH (1 mL) was added HBr/AcOH (5.5 M, 1 mL) at 30° C. under N₂.The mixture was stirred at 30° C. under N₂ for another 0.5 h. LC-MSindicated the reaction completed. Water (20 mL) was added and extractedwith MTBE (5 mL×2). Then the aqueous layer was lyophilized to give acrude, which was purified by prep-HPLC (CH₃CN/H₂O/TFA) to give(3S)—N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoyl)piperidine-3-carboxamide(3; 15 mg, 29.61 μmol, 8.62% yield) as a light yellow solid. MSm/z=507.2 (MH⁺).

Example 11. Preparation of(3R)—N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoyl)piperidine-3-carboxamide(4)

Tert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(4c)

To a solution of (3R)-1-tert-butoxycarbonylpiperidine-3-carboxylic acid(190 mg, 828.72 μmol, 1.20 eq) in DMF (10 mL) were added HATU (320 mg,841.60 μmol, 1.22 eq) and DIPEA (267 mg, 2.07 mmol, 2.99 eq) under 0° C.After stirring for 0.5 h under 0° C.,benzyl-N-[(5S)-5-(amino-chloranyl)-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the resultingmixture was stirred at 30° C. for 3 h. TLC (PE:EA=3:1) indicated thereaction completed. The mixture was diluted with H₂O (15 mL×2) andextracted with EA (20 mL×2). The combined organic layers were washedwith H₂O (20 mL×2), dried over Na₂SO₄, filtered and concentrated to givetert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(4c; 600 mg, crude) as a oil, which was used directly for next stepwithout further purification.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamate (4b)

To a solution oftert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(600 mg, 985.63 μmol, 1.00 eq) in EA (10 mL) was added HO/EA (4 M, 4 mL,16.23 eq). The reaction mixture was stirred at 30° C. for 1 h. Thesuspension was filtered and the filter cake was collected to affordbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamatehydrochloride (4b; 700 mg, crude), which was used directly for next stepwithout purification. It was confirmed by LC-MS.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoyl)piperidine-3-carbonyl]amino]hexyl]carbamate(4a)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamate;hydrochloride (700 mg, 1.28 mmol, 1.00 eq) in DCM (10 mL) was added TEA(2.19 g, 21.64 mmol, 16.91 eq). The mixture was cooled to 0° C., andthen 3-phenylpropanoyl chloride (1.14 g, 6.76 mmol, 5.28 eq) was addeddropwise. The resulting mixture was stirred at 0° C. for 30 min. LC-MSindicated the reaction completed. The reaction was quenched by H₂O (20mL) and extracted with DCM (20 mL×2). The combined organic layers weredried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoy)piperidine-3-carbonyl]amino]hexyl]carbamate(4a; 600 mg, crude), which was used directly for next step withoutpurification.

(3R)—N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoyl)piperidine-3-carboxamide(4)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoy)piperidine-3-carbonyl]amino]hexyl]carbamate(600 mg, 936.34 μmol, 1.00 eq) in AcOH (10 mL) was added HBr/AcOH(936.34 μmol, 1.00 eq) (2 mL, 33% purity) under N₂. The reaction mixturewas stirred at 32° C. for 30 min. LC-MS indicated the desired productwas detected. The mixture was diluted with H₂O (100 mL) and MeOH (20mL), washed with MTBE (50 mL×2). The resulting solution was lyophilizedto give a residue, which was purified by prep-HPLC (mobile phase: CH₃CN,H₂O/TFA) to give(3R)—N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoyl)piperidine-3-carboxamide;2,2,2-trifluoroacetic acid trifluoroacetate (4; 30 mg, 40.83 μmol, 4.36%yield) as a yellow solid. MS m/z=507.2 (MH⁺).

Example 12. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-2-(trifluoromethyl)benzamide(7)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[2-(trifluoromethyl)benzoyl]amino]hexyl]carbamate(7a)

To a mixture ofbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(400 mg, 921.77 μmol, 1.00 eq), 2-(trifluoromethyl)benzoic acid (210 mg,1.11 mmol, 1.20 eq), HOBt (162 mg, 1.20 mmol, 1.30 eq), EDCI (230 mg,1.20 mmol, 1.30 eq) in DCM (10 mL) was added DIPEA (358 mg, 2.77 mmol,3.00 eq) at 0° C. Then the mixture was stirred at 30° C. for 16 h. LC-MSindicated the reaction completed. The mixture was washed with water (5mL×3), dried over Na₂SO₄, concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[2-(trifluoromethyl)benzoyl]amino]hexyl]carbamate(7a; 500 mg, crude) as a yellow solid.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-2-(trifluoromethyl)benzamide (7)

To a mixture ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[2-(trifluoromethyl)benzoyl]amino]hexyl]carbamate (300 mg, 526.69 μmol, 1.00 eq) in AcOH (1mL) was added HBr/AcOH (5.5 M, 1 mL). The mixture was stirred at 30° C.under N₂ for 0.5 h. LC-MS indicated the reaction completed. Water (20mL) was added and extracted with MTBE (5 mL×2). Then the aqueous layerwas lyophilized to give a crude, which was purified by prep-HPLC(CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-2-(trifluoromethyl)benzamidetrifluoroacetate (7; 20 mg, 36.40 μmol, 6.9% yield) as a light yellowsolid. MS m/z=437.0 (MH⁺).

Example 13. Preparation of(3S)-1-acetyl-N-(6-amino-1-(benzo[d]thiazol-2-yl)-1-oxohexan-2-yl)piperidine-3-carboxamide(8 and 10)

Tert-butyl-(3S)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(8c)

A mixture of (3S)-1-tert-butoxycarbonylpiperidine-3-carboxylic acid (693mg, 3.02 mmol, 1.20 eq), HATU (1.15 g, 3.02 mmol, 1.20 eq), DIPEA (977mg, 7.56 mmol, 3.00 eq) in DMF (20 mL) was stirred at 0° C. for 1 h.Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (1.00 g, 2.52 mmol, 1.00 eq) was added and the mixture wasstirred at 30° C. for 16 h. TLC (PE:EA=3:1) indicated the reactioncompleted. EA (100 mL) was added and washed with water (50 mL×3), driedover Na₂SO₄, concentrated to givetert-butyl-(3S)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(8c; 1.10 g, 1.81 mmol, 71.7% yield) as a yellow solid.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-piperidine-3-carbonyl]amino]hexyl]carbamate(8b)

To a mixture oftert-butyl-(3S)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(1.10 g, 1.81 mmol, 1.00 eq) in EA (10 mL) was added HO/EA (4 M, 2 mL)and the reaction mixture was stirred at 30° C. for 2 h. TLC (PE:EA=3:1)indicated the reaction completed and the reaction was filtered to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-piperidine-3-carbonyl]amino]hexyl]carbamatehydrochloride (8b; 700 mg, crude) as a light yellow solid.

Benzyl-N-[(5S)-5-[[(3S)-1-acetylpiperidine-3-carbonyl]amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(8a)

To a mixture ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3S)-piperidine-3-carbonyl]amino]hexyl]carbamatehydrochloride (700 mg, 1.28 mmol, 1.00 eq), TEA (390 mg, 3.85 mmol, 3.00eq) in DCM (5 mL) was added acetyl chloride (150 mg, 1.93 mmol, 1.50 eq)dropwise at 0° C. under N₂ and the reaction mixture was stirred 0° C.under N₂ for 1 h. TLC (PE:EA=3:1) indicated the reaction completed, thenDCM (20 mL) was added and the mixture was washed with water (10 mL×3),dried over Na₂SO₄, concentrated to givebenzyl-N-[(55)-5-[[(35)-1-acetylpiperidine-3-carbonyl]amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(8a; 800 mg, crude) as a yellow oil.

(3S)-1-acetyl-N-(6-amino-1-(benzo[d]thiazol-2-yl)-1-oxohexan-2-yl)piperidine-3-carboxamide(8 and 10)

To a mixture ofbenzyl-N-[(5S)-5-[[(3S)-1-acetylpiperidine-3-carbonyl]amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(800 mg, 1.45 mmol, 1.00 eq) in AcOH (5 mL) was added HBr/AcOH (5.5 M, 2mL). Then the reaction mixture was stirred at 30° C. for 0.5 hr. LC-MSindicated the reaction completed. Water (30 mL) was added and extractedwith MTBE (10 mL×2), the aqueous layer was lyophilized to give a crude,which was purified by prep-HPLC (CH₃CN/H₂O/TFA) to give(3S)-1-acetyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]piperidine-3-carboxamidetrifluoroacetate (8; 21.20 mg, 39.96 μmol, 2.8% yield) and(3S)-1-acetyl-N-[(1R)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]piperidine-3-carboxamidetrifluoroacetate (10; 24.20 mg, 45.61 μmol, 3.2% yield) as light yellowsolids. MS m/z=417.1 (MH⁺).

Example 14. Preparation of(3R)-1-acetyl-N-(6-amino-1-(benzo[d]thiazol-2-yl)-1-oxohexan-2-yl)piperidine-3-carboxamide(9 and 11)

Tert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(9c)

To a solution of (3R)-1-tert-butoxycarbonylpiperidine-3-carboxylic acid(633 mg, 2.76 mmol, 1.20 eq) in DMF (10 mL) were added HATU (2.62 g,6.90 mmol, 3.00 eq) and DIPEA (892 mg, 6.90 mmol, 3.00 eq) under 0° C.After being stirred for 0.5 h under 0° C.,benzyl-N-[(5S)-5-(amino-chloranyl)-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (1.00 g, 2.30 mmol, 1.00 eq) was added and the resultingmixture was stirred at 30° C. for 6 h. TLC (PE:EA=3:1) indicated thereaction completed. The mixture was diluted with H₂O (15 mL×2) andextracted with EA (20 mL×2). The combined organic layers were washedwith H₂O (20 mL×2), dried over Na₂SO₄, filtered and concentrated to givetert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(9c; 1.40 g, crude) as a oil, which was used directly for next stepwithout further purification.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamate(9b)

To a solution oftert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(821.36 μmol, 1.00 eq) (1.4 g, crude) in EA (20.00 mL) was added HO/EA(4 M, 600.00 uL, 2.92 eq). The reaction mixture was stirred at 30° C.for 4 h. The suspension was filtered and the filter cake was collectedto affordbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamate hydrochloride (9b; 2.20 g, crude), which was useddirectly for next step without purification.

Benzyl-N-[(5S)-5-[[(3R)-1-acetylpiperidine-3-carbonyl]amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(9a)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamatehydrochloride (4.04 mmol, 1.00 eq) (2.2 g, crude) in DCM (20 mL) wasadded TEA (1.23 g, 12.11 mmol, 3.00 eq). The mixture was cooled to 0°C., and then acetyl chloride (475 mg, 6.05 mmol, 1.50 eq) was addeddropwise. The resulting mixture was stirred at 0° C. for 30 min. LC-MSindicated the reaction completed. The reaction was quenched by H₂O (30mL) and extracted with DCM (20 mL×2). The combined organic layers weredried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-5-[[(3R)-1-acetylpiperidine-3-carbonyl]amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(9a; 1.50 g, crude), which was used directly for next step withoutpurification.

(3R)-1-acetyl-N-(6-amino-1-(benzo[d]thiazol-2-yl)-1-oxohexan-2-yl)piperidine-3-carboxamide(9 and 11)

To a solution ofbenzyl-N-[5-[[(3R)-1-acetylpiperidine-3-carbonyl]amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(2.30 mmol, 1.00 eq) (1.5 g, crude) in AcOH (10 mL) was added HBr/AcOH(2.30 mmol, 1.00 eq) (2 mL, 33% purity) under N₂. The reaction mixturewas stirred at 32° C. for 30 min. LC-MS indicated the desired productwas detected. The mixture was diluted with H₂O (100 mL) and MeOH (20mL), washed with MTBE (50 mL×2). The resulting solution was underlyophilized to give a residue, which was purified by prep-HPLC (mobilephase: CH₃CN/H₂O/TFA) to give(3R)-1-acetyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]piperidine-3-carboxamidetrifluoroacetate (9; 5.30 mg, 9.99 μmol, 0.4% yield) (yield over 4steps) and(3R)-1-acetyl-N-[(1R)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]piperidine-3-carboxamidetrifluoroacetate (11; 49.70 mg, 119.32 μmol, 5.2% yield) (yield over 4steps) as yellow solids. MS m/z=417.1 (MH⁺).

Example 15. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-2-carboxamide(12)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-2-carbonylamino)hexyl]carbamate(12a)

To a solution of pyridine-2-carboxylic acid (102 mg, 829.58 μmol, 1.20eq) in THF (8 mL) were added HATU (315 mg, 829.58 μmol, 1.20 eq) andDIPEA (268 mg, 2.07 mmol, 3.00 eq) at 0° C., the resulting mixture wasstirred at 0° C. for 15 min. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added at 0° C., thereaction mixture was stirred at 30° C. for another 3 h. LC-MS indicatedthe starting material was consumed completely. The reaction was quenchedby H₂O (30 mL) and then extracted with EA (20 mL×2), the combinedorganic phase was washed with saturated brine (30 mL) and concentratedto givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-2-carbonylamino)hexyl]carbamate(12a; 698 mg, crude) as a yellow solid.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-2-carboxamide(12)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-2-carbonylamino)hexyl]carbamate(682 mg, 1.36 mmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (1 mL,33% purity) at 20° C., the reaction mixture was stirred at 20° C. for 1h. LC-MS indicated the starting material was consumed and desiredcompound was detected. After stirring for another 30 min, the reactionwas quenched by H₂O (10 mL) and washed with MTBE (20 mL). The organicphase was extracted with H₂O (20 mL×2) and the aqueous phases werecombined and lyophilized to give a crude, which was purified byprep-HPLC (CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-2-carboxamidetrifluoroacetate (12; 67.44 mg, 139.78 μmol, 10.3% yield, 74.4% ee) as ayellow solid. MS m/z=369.1 (MH⁺).

Example 16. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-4-carboxamide(13)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-4-carbonylamino)hexyl]carbamate(13a)

To a solution of isonicotinic acid (102 mg, 829.58 μmol, 1.20 eq) in THF(8 mL) were added DIPEA (268 mg, 2.07 mmol, 3.00 eq) and HATU (315.43mg, 829.58 μmol, 1.20 eq) at 0° C., the reaction mixture was stirred at0° C. for 15 min. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added at 0° C., thereaction mixture was stirred at 30° C. for another 3 h. LC-MS indicatedthe starting material was consumed completely. The reaction was quenchedby H₂O (30 mL) and then extracted with EA (20 mL×2), the combinedorganic phase was washed with saturated brine (30 mL) and concentratedto givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-4-carbonylamino)hexyl]carbamate(13a; 727 mg, crude) as a red solid, which was used directly withoutfurther purification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-4-carboxamide(13)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-4-carbonylamino)hexyl]carbamate(727 mg, 1.45 mmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (1 mL,33% purity). The reaction mixture was stirred at 20° C. for 3 h. LC-MSindicated the starting material was consumed completely. The reactionwas quenched by H₂O (10 mL) and washed with MTBE (20 mL). The organicphase was extracted with H₂O (20 mL×2) and the aqueous phases werecombined and lyophilized to give a crude, which was purified byprep-HPLC (CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-4-carboxamide trifluoroacetate (13; 75.78 mg, 157.07μmol, 10.8% yield, 69.3% ee) as a yellow solid. MS m/z=369.0 (MH⁺).

Example 17. Preparation of(3R)—N-[5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoyl)piperidine-3-carboxamide(14/15)

Tert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(14c)

To a solution of (3R)-1-tert-butoxycarbonylpiperidine-3-carboxylic acid(3.17 g, 13.83 mmol, 1.20 eq) in DMF (60 mL) were added HATU (5.34 g,14.06 mmol, 1.22 eq) and DIPEA (4.45 g, 34.45 mmol, 2.99 eq) under 0° C.After stirring for 0.5 h under 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (5.00 g, 11.52 mmol, 1.00 eq) was added and the resultingmixture was stirred at 30° C. for 3 h. TLC (PE:EA=3:1) indicated thereaction completed. The mixture was diluted with H₂O (80 mL×2) andextracted with EA (100 mL×2). The combined organic layers were washedwith H₂O (60 mL×2), dried over Na₂SO₄, filtered and concentrated to givetert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(9.80 g, crude) as a oil, which was used directly for next step withoutfurther purification.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamate(14b)

To a solution oftert-butyl-(3R)-3-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]piperidine-1-carboxylate(16.10 mmol, 1.00 eq) (9.8 g, crude) in EA (100 mL) was added HCl/EA (4M, 20 mL, 4.97 eq). The reaction mixture was stirred at 30° C. for 1 h.The suspension was filtered and the filter cake was collected to affordbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamatehydrochloride (10.90 g, crude), which was used directly for next stepwithout purification.

Benzyl-N-[6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoyl)piperidine-3-carbonyl]amino]hexyl]carbamate(14a)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-piperidine-3-carbonyl]amino]hexyl]carbamate;hydrochloride (20 mmol, 1.00 eq) (10.9 g, crude) in DCM (100 mL) wasadded TEA (6.07 g, 60.00 mmol, 3.00 eq). The mixture was cooled to 0°C., and then 3-phenylpropanoyl chloride (5.06 g, 30.00 mmol, 1.50 eq)was added dropwise. The resulting mixture was stirred at 0° C. for 30min. LC-MS indicated the reaction completed. The reaction was quenchedby H₂O (60 mL) and extracted with DCM (100 mL×2). The combined organiclayers were dried over Na₂SO₄, filtered and concentrated to give acrude, which was purified by column chromatography (PE:EA=8:1 to 3:1) togivebenzyl-N-[6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoy)piperidine-3-carbonyl]amino]hexyl]carbamate(2.77 g, 4.32 mmol, 21.6% yield) as a yellow solid.

Benzyl-N-[6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoy)piperidine-3-carbonyl]amino]hexyl]carbamate(1 g) was purified by chiral SFC separation (Chiral SFC separationmethod: Instrument: SFC 80, Column: AD-10 μm, Mobile phase: A for CO₂and B for EtOH (0.1% NH₃.H₂O), Gradient: B 50%, Flow rate: 70 mL/min,Back pressure: 100 bar, Column temperature: 35° C., Wavelength: 220 nm)to give products (peak 1: 0.4 g) as a yellow solid and (peak 2: 0.51 g)as brown oil.

(3R)—N-[5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoyl)piperidine-3-carboxamide;2,2,2-trifluoroacetic acid (14/15)

To a solution ofbenzyl-N-[6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoyl)piperidine-3-carbonyl]amino]hexyl]carbamate (400 mg, 624.23 μmol, 1.00eq) (Peak 1) in AcOH (6 mL) was added HBr/AcOH (624.23 μmol, 1.00 eq) (2mL, 33% purity) under N₂. The reaction mixture was stirred at 30° C. for1 hr. LC-MS indicated the desired product was detected. The mixture wasdiluted with H₂O (100 mL) and MeOH (20 mL), washed with MTBE (50 mL×2).The resulting solution was lyophilized to give a residue, which waspurified by prep-HPLC (mobile phase: CH₃CN/H₂O/TFA) to give(3R)—N-[5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoy)piperidine-3-carboxamidetrifluoroacetate (14; 39 mg, 76.97 μmol, 12.3% yield) as a light yellowsolid. MS m/z=507.2 (MH⁺).

To a solution ofbenzyl-N-[6-(1,3-benzothiazol-2-yl)-6-oxo-5-[[(3R)-1-(3-phenylpropanoyl)piperidine-3-carbonyl]amino]hexyl]carbamate (510 mg, 795.89 μmol, 1.00eq) (Peak 2) in AcOH (6 mL) was added HBr/AcOH (795.89 μmol, 1.00 eq) (2mL, 33% purity) under N₂. The reaction mixture was stirred at 30° C. for1 hr. LC-MS indicated the desired product was detected. The mixture wasdiluted with H₂O (100 mL) and MeOH (20 mL), washed with MTBE (50 mL×2).The resulting solution was lyophilized to give a residue, which waspurified by prep-HPLC (mobile phase: CH₃CN/H₂O/TFA) to give(3R)—N-[5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-(3-phenylpropanoy)piperidine-3-carboxamidetrifluoroacetate (15; 43 mg, 84.87 μmol, 10.7% yield) as yellow oil. MSm/z=507.2 (MH⁺).

Example 18. Preparation ofN-[(14)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-3-carboxamide(18/19)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-3-carbonylamino)hexyl]carbamate(18a)

To a solution of tetrahydropyran-3-carboxylic acid (108 mg, 829.58 μmol,1.20 eq) in THF (5 mL) were added HATU (390 mg, 1.03 mmol, 1.48 eq) andDIPEA (270 mg, 2.09 mmol, 3.02 eq) under 0° C. After being stirred for0.5 h under 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the resultingmixture was stirred at 28° C. for 4.5 h. LC-MS indicated the reactioncompleted. The mixture was diluted with H₂O (15 mL×2) and extracted withEA (20 mL×2). The combined organic layers were washed with H₂O (20 mL),dried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-3-carbonylamino)hexyl]carbamate(700 mg, crude) as a yellow solid. It was used directly for next stepwithout further purification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-3-carboxamide(18/19)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-3-carbonylamino)hexyl]carbamate(1.37 mmol, 1.00 eq) (0.7 g, crude) in AcOH (6 mL) was added HBr/AcOH(1.37 mmol, 1.00 eq) (2 mL, 33% purity) under N₂. The reaction mixturewas stirred at 28° C. for 1 hr. LC-MS indicated the desired product wasdetected. The mixture was diluted with H₂O (10 mL) and MeOH (2 mL),washed with MTBE (10 mL×2). The resulting solution was diluted with H₂Oand then lyophilized to give a residue, which was purified by prep-HPLC(mobile phase: CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-3-carboxamidetrifluoroacetates (14 mg Peak 1, compound 18) and (15 mg Peak 2,compound 19) as yellow solids. MS m/z=376.1 (MH⁺).

Example 19. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-4-carboxamide(22)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-4-carbonylamino)hexyl]carbamate(22a)

To a solution of tetrahydropyran-4-carboxylic acid (108 mg, 829.58 μmol,1.20 eq) in THF (5 mL) were added HATU (390 mg, 1.03 mmol, 1.48 eq) andDIPEA (270 mg, 2.09 mmol, 3.02 eq) under 0° C. After being stirred for0.5 h under 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the resultingmixture was stirred at 28° C. for 4.5 h. LC-MS indicated the reactioncompleted. The mixture was diluted with H₂O (15 mL×2) and extracted withEA (20 mL×2). The combined organic layers were washed with H₂O (20 mL),dried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-4-carbonylamino)hexyl]carbamate(680 mg, crude) as a light yellow solid. It was used directly for nextstep without further purification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-4-carboxamide(22)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-4-carbonylamino)hexyl]carbamate(1.33 mmol, 1.00 eq) (0.68 g, crude) in AcOH (5 mL) was added HBr/AcOH(1.33 mmol, 1.00 eq) (2 mL, 33% purity) under Na. The reaction mixturewas stirred at 30° C. for 30 min. LC-MS indicated the desired productwas detected. The mixture was diluted with H₂O (10 mL) and MeOH (2 mL),washed with MTBE (10 mL×2). The resulting solution was lyophilized togive a residue, which was purified by prep-HPLC (mobile phase:CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-4-carboxamidetrifluoroacetate (45 mg) as a yellow solid. MS m/z=376.1 (MH⁺).

Example 20. Preparation ofBenzyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl) pentyl]carbamate(23)

Tert-butyl-N-[(5S)-5-(benzyloxycarbonylamino)-6-hydroxy-hexyl]carbamate(23d)

To a solution of(2S)-2-(benzyloxycarbonylamino)-6-(tert-butoxycarbonylamino)-hexanoicacid (25.00 g, 65.72 mmol, 1.00 eq) in THF (250 mL) was added TEA (6.65g, 65.72 mmol, 1.00 eq). The solution was cooled to −10° C. and thendropwise addition of iso-butylchloroformate (9.87 g, 72.27 mmol, 1.10eq) (It has suspensoid appeared). The resulting suspension was stirredfor 2 h at 0° C. The reaction mixture was filtered and cooled to −10° C.NaBH₄ (5.22 g, 138.00 mmol, 2.10 eq) was dissolved in water (50 mL) at0° C. and the solution was added dropwise to the THF solution (heavy CO₂evolution). The reaction mixture was allowed to warm to 30° C. andstirred for 3 hr. The reaction mixture was acidified with 1M HClsolution, and the aqueous phase was extracted with EA (400 mL×2). Thecombined organic layers were washed with water, saturated aqueous NaHCO₃solution and brine; dried over Na₂SO₄, concentrated to give a residue,which was purified by flash column chromatography (PE:EA=2:1 to EA) toaffordtert-butyl-N-[(5S)-5-(benzyloxycarbonylamino)-6-hydroxy-hexyl]carbamate(19.30 g, 52.67 mmol, 80.1% yield) as a oil.

Tert-butyl-N-[(5S)-5-(benzyloxycarbonylamino)-6-oxo-hexyl]carbamate(23c)

A solution oftert-butyl-N-[(5S)-5-(benzyloxycarbonylamino)-6-hydroxy-hexyl]carbamate(19.20 g, 52.39 mmol, 1.00 eq) in DCM (200 mL) was cooled to 0° C. ThenDess-Martin Periodinane (22.22 g, 52.39 mmol, 1.00 eq) was added inportions. The suspension was allowed to warm to room temperature (30°C.) and stirred 14 hr. TLC indicated the starting material was remaineda little. A (100 mL: 100 mL) mixture of saturated aqueous NaHCO₃solution and a 1M Na₂S₂O₃ solution was added and the resulting biphasicsystem was stirred vigorously for 30 min. The organic layer wasseparated and the aqueous layer was extracted with DCM (200 mL). Thecombined organic layers were distilled in vacuo and the resulting oilwere taken up in EA (100 mL) and washed four times with theNaHCO₃/Na₂S₂O₃ mixture, water and brine, dried over Na₂SO₄ andconcentrated in vacuo to give crudetert-butyl-N-[(5S)-5-(benzyloxycarbonylamino)-6-oxo-hexyl]carbamate(17.33 g, crude) as a yellowish oil. It was directly used in the nextstep without further purification.

Tert-butyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(benzyloxycarbonylamino)-6-hydroxy-hexyl]carbamate(23b)

A solution of 1,3-benzothiazole (5.56 g, 41.16 mmol, 3.00 eq) in THF(100 mL) was cooled to −78° C. Then n-BuLi (2.5 M, 16.5 mL, 3.01 eq)(2.5 M in THF, 16.5 mL) was added dropwise at −78° C. under N₂. Afterbeing stirred for 1 h at −78° C.,tert-butyl-N-[(5S)-5-(benzyloxycarbonylamino)-6-oxo-hexyl]carbamate(13.72 mmol, 1.00 eq) (5 g, crude) in THF was added dropwise at −78° C.The resulting mixture was stirred for another 3 h at −78° C. LC-MSindicated the starting material disappeared and the desired product wasdetected. The reaction was quenched by NH₄Cl (60 mL) and extracted withEA (100 mL). The organic layer was separated, washed with brine, driedover MgSO₄, filtered and concentrated to give a crude product, which waspurified by flash column chromatography (PE:EA=5:1 to 3:1) to givetert-butyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(benzyloxycarbonylamino)-6-hydroxy-hexyl]carbamate(1.77 g, 3.54 mmol, 25.8% yield) as a yellow oil.

Benzyl-N-[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(tert-butoxycarbonyl-amino)pentyl]carbamate(23a)

A solution oftert-butyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(benzyloxycarbonylamino)-6-hydroxy-hexyl]carbamate(500 mg, 1.00 mmol, 1.00 eq) in DCM (6 mL) was cooled to 0° C. ThenDess-Martin (430 mg, 1.01 mmol, 1.01 eq) was added in portions. Thesuspension was allowed to warm to room temperature (28° C.) and stirred14 hr. TLC indicated the starting material was consumed completely. A(10 mL: 10 mL) mixture of saturated aqueous NaHCO₃ solution and a 1MNa₂S₂O₃ solution was added and the resulting biphasic system was stirredvigorously for 30 min. The organic layer was separated and the aqueouslayer was extracted with DCM (20 mL). The combined organic layers weredistilled in vacuo and the resulting oil were taken up in EA (20 mL) andwashed four times with the NaHCO₃/Na₂S₂O₃ mixture, water and brine,dried over Na₂SO₄ and concentrated in vacuo to give a crude, which waspurified by flash chromatography to givebenzyl-N-[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(tert-butoxycarbonyl-amino)pentyl]carbamate(200 mg, 401.92 μmol, 40.2% yield) as a yellow solid.

Benzyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]carbamate(23)

To a solution ofbenzyl-N-[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(tert-butoxycarbonylamino)pentyl]carbamate (200 mg, 401.92 μmol, 1.00 eq) in EA (6 mL) was addedHCl/EA (4 M, 4 mL, 39.81 eq). The resulting mixture was stirred at 28°C. for 14 hr. It was filtered and the filter cake was diluted withdeionized water. The solution was lyophilized to givebenzyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]carbamatehydrochloride (43.9 mg) as a yellow solid. MS m/z=398.1 (MH⁺).

Example 21. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]cyclo-pentanecarboxamide(24)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(cyclopentanecarbonylamino)-6-oxo-hexyl]carbamate(24a)

To a mixture ofbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq), cyclopentanecarboxylicacid (79 mg, 691.32 μmol, 1.00 eq), DIPEA (268 mg, 2.07 mmol, 3.00 eq)in THF (5 mL) was added HATU (315 mg, 829.58 μmol, 1.20 eq). The mixturewas stirred at 30° C. for 16 h. LC-MS indicated the starting materialwas consumed completely. EA(50 mL) was added and the mixture was washedwith water (20 mL×2), dried over Na₂SO₄, concentrated to give a crude,which was purified by column chromatography on silica gel (PE:EA=5: 1)to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(cyclopentanecarbonylamino)-6-oxo-hexyl]carbamate(280 mg, 567.24 μmol, 82.1% yield) as a yellow solid.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]cyclo-pentanecarboxamide(24)

To a mixture ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(cyclopentanecarbonylamino)-6-oxo-hexyl]carbamate(280 mg, 567.24 μmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (5.5 M,1 mL). The mixture was stirred at 30° C. for 1 h. LC-MS indicated thestarting material was consumed completely. Water (50 mL) was added andextracted with MTBE (10 mL×2). The aqueous layer was lyophilized to givea crude, which was purified by prep-HPLC (CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]cyclo-pentanecarboxamidetrifluoroacetate (21.63 mg, 60.17 μmol, 10.6% yield) as a white solid.MS m/z=360 (MH⁺). ¹H NMR (CD₃OD, 400 MHz) d 8.22 (d, J=7.6, 1H), 8.15(d, J=7.6, 1H), 7.69-7.61 (m, 2H), 5.68 (dd, J=9.6, J=4.0, 1H),3.07-2.92 (m, 2H), 2.85-2.75 (m, 1H), 2.22-2.12 (m, 1H), 1.95-1.55 (m,13H).

Example 22. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-2-carboxamide(25/26)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-2-carbonylamino)hexyl]carbamate(2)

To a solution of tetrahydropyran-2-carboxylic acid (108 mg, 829.88 μmol,1.20 eq) in THF (5 mL) were added HATU (395 mg, 1.04 mmol, 1.50 eq) andDIPEA (270 mg, 2.09 mmol, 3.02 eq) under 0° C. After being stirred for0.5 h under 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the resultingmixture was stirred at 28° C. for 4.5 h. LC-MS indicated the reactioncompleted. The mixture was diluted with H₂O (10 mL) and extracted withEA (20 mL×2). The combined organic layers were washed with H₂O (20 mL),dried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-2-carbonylamino)hexyl]carbamate(950 mg, crude) as a yellow oil. It was used directly for next stepwithout further purification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-2-carboxamide(152/153)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydropyran-2-carbonyl-amino)hexyl]carbamate(1.86 mmol, 1.00 eq) (0.95 g, crude) in AcOH (6 mL) was added HBr/AcOH(1.86 mmol, 1.00 eq) (2 mL, 33% purity) under N₂. The reaction mixturewas stirred at 30° C. for 1 hr. LC-MS indicated the desired product wasdetected. The mixture was diluted with H₂O (10 mL) and MeOH (2 mL),washed with MTBE (10 mL×2). The resulting solution was lyophilized togive a residue, which was purified by prep-HPLC (mobile phase:CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydropyran-2-carboxamidetrifluoroacetates (15 mg; Peak 1, 25) and (13 mg; Peak 2, 26) as yellowsolids. MS m/z=376.1 (MH⁺).

Example 23. Preparation of4-acetyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]morpholine-2-carboxamide(29)

Tert-butyl-2-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonyl-amino)pentyl]carbamoyl]morpholine-4-carboxylate(29c)

To mixture of 4-tert-butoxycarbonylmorpholine-2-carboxylic acid (320 mg,1.38 mmol, 1.20 eq), DIPEA (446 mg, 3.45 mmol, 3.00 eq) in THF (10 mL)was added HATU (525 mg, 1.38 mmol, 1.20 eq) at 0° C. and the mixture wasstirred at 0° C. for 0.5 h. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (500 mg, 1.15 mmol, 1.00 eq) was added and the mixture wasstirred at 30° C. for 3 h. LC-MS indicated the starting material wasconsumed completely. EA (30 mL) was added and the mixture was washedwith water (10 mL×2), dried over Na₂SO₄, concentrated to give a crude,which was purified by column chromatography on silica gel (PE:EA=3: 1 toPE:EA=1: 1) to givetert-butyl-2-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]morpholine-4-carboxylate(600 mg, crude) as a yellow solid.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(morpholine-2-carbonylamino)-6-oxo-hexyl]carbamate(29b)

To a mixture oftert-butyl-2-[[(1S)-1-(1,3-benzothiazole-2-carbonyl)-5-(benzyloxycarbonylamino)pentyl]carbamoyl]morpholine-4-carboxylate(600 mg, 982.45 μmol, 1.00 eq) in EA (5 mL) was added HO/EA (4 M, 1 mL).The mixture was stirred at 30° C. for 2 hr. TLC (PE:EA=1:1) indicatedthe starting material was consumed completely. The mixture was filteredto givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(morpholine-2-carbonylamino)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 548.39 μmol, 55.8% yield) as a light yellowsolid.

Benzyl-N-[(5S)-5-[(4-acetylmorpholine-2-carbonyl)amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(29a)

To a mixture ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-(morpholine-2-carbonylamino)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 548.39 μmol, 1.00 eq), TEA (166 mg, 1.65 mmol,3.00 eq) in DCM (5 mL) was added acetyl chloride (86 mg, 1.10 mmol, 2.00eq) dropwise at 0° C. under N₂. The mixture was stirred at 0° C. underN₂ for 10 min. LC-MS indicated the starting material was consumedcompletely. DCM (20 mL) was added and the mixture was washed with water(10 mL×3), dried over Na₂SO₄, concentrated to givebenzyl-N-[(5S)-5-[(4-acetylmorpholine-2-carbonyl)amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(200 mg, 361.90 μmol, 67.0% yield) as a yellow solid.

4-acetyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]morpholine-2-carboxamide(29)

To a mixturebenzyl-N-[(55)-5-[(4-acetylmorpholine-2-carbonyl)amino]-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamate(200 mg, 361.90 μmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (5.5 M,1 mL). The mixture was stirred at 30° C. for 1 h. LC-MS indicated thestarting material was consumed completely. Water (40 mL) was added andthe mixture was extracted with MTBE (10 mL×2). The aqueous layer waslyophilized to give a crude, which was purified by prep-HPLC(CH₃CN/H₂O/TFA) to give4-acetyl-N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]morpholine-2-carboxamidetrifluoroacetate (14.46 mg, 27.15 μmol, 7.5% yield) as a white solid. MSm/z=419.1 (MH⁺).

Example 24. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-3-carboxamide(30)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-3-carbonylamino)hexyl]carbamate(30a)

To a solution of nicotinic acid (102 mg, 829.58 μmol, 1.20 eq) in THF (8mL) were added DIPEA (268 mg, 2.07 mmol, 3.00 eq) and HATU (315 mg,829.58 μmol, 1.20 eq) at 0° C., the reaction mixture was stirred at 0°C. for 15 min. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added at 0° C., thereaction mixture was stirred at 30° C. for another 3 h. LC-MS indicatedthe starting material was consumed completely. The reaction was quenchedby H₂O (30 mL) and then extracted with EA (20 mL×2), the organic phaseswere combined, washed with saturated brine (30 mL) and concentrated togivebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-3-carbonylamino)hexyl]carbamate(638 mg, crude) as a red solid, which was used directly without furtherpurification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-3-carboxamide(30)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(pyridine-3-carbonylamino)hexyl]carbamate(638 mg, 1.27 mmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (1 mL,33% purity). The reaction mixture was stirred at 30° C. for 1 h. LC-MSindicated the starting material was consumed completely. The reactionwas quenched by H₂O (10 mL) and washed with MTBE (20 mL). The organicphase was extracted with H₂O (20 mL×2) and the aqueous phases werecombined and lyophilized to give a crude product, which was purified byprep-HPLC (CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]pyridine-3-carboxamidetrifluoroacetate (50.96 mg, 105.62 μmol, 8.3% yield, 38.2% ee) as ayellow solid. MS m/z=369.1 (MH⁺).

Example 25. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetralin-1-carboxamide(31)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetralin-1-carbonylamino)hexyl]carbamate(31a)

To a solution of tetralin-1-carboxylic acid (150 mg, 851.26 μmol, 1.23eq) in THF (5 mL) were added HATU (390 mg, 1.03 mmol, 1.48 eq) and DIPEA(270 mg, 2.09 mmol, 3.02 eq) under 0° C. After being stirred for 0.5 hunder 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the resultingmixture was stirred at 28° C. for 5.5 h. LC-MS indicated the reactioncompleted. The mixture was diluted with H₂O (10 mL) and extracted withEA (20 mL×2). The combined organic layers were washed with H₂O (20 mL),dried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetralin-1-carbonylamino)hexyl]carbamate(660.00 mg, crude) as a yellow oil. It was used directly for next stepwithout further purification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetralin-1-carboxamide(31)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetralin-1-carbonylamino)hexyl]carbamate(1.19 mmol, 1.00 eq) (0.66 g, crude) in AcOH (6 mL) was added HBr/AcOH(1.19 mmol, 1.00 eq) (2 mL, 33% purity) under N₂. The reaction mixturewas stirred at 28° C. for 1 hr. LC-MS indicated the desired product wasdetected. The mixture was diluted with H₂O (10 mL) and MeOH (2 mL),washed with MTBE (10 mL×2). The resulting solution was diluted with H₂Oand then lyophilized to give a residue, which was purified by prep-HPLC(CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetralin-1-carboxamidetrifluoroacetate (70 mg) as a yellow solid. MS m/z=422.1 (MH⁺).

Example 26. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetralin-2-carboxamide(32)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetralin-2-carbonylamino)hexyl]carbamate(32a)

To a solution of tetralin-2-carboxylic acid (150 mg, 851.26 μmol, 1.23eq) in THF (5 mL) were added HATU (390 mg, 1.03 mmol, 1.48 eq) and DIPEA(270 mg, 2.09 mmol, 3.02 eq) under 0° C. After being stirred for 0.5 hunder 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the resultingmixture was stirred at 28° C. for 5.5 h. LC-MS indicated the reactioncompleted. The mixture was diluted with H₂O (15 mL×2) and extracted withEA (20 mL×2). The combined organic layers were washed with H₂O (20 mL),dried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetralin-2-carbonylamino)hexyl]carbamate(700 mg, crude) as a yellow solid. It was used directly for next stepwithout further purification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetralin-2-carboxamide(32)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetralin-2-carbonylamino)hexyl]carbamate(1.26 mmol, 1.00 eq) (0.7 g, crude) in AcOH (5 mL) was added HBr/AcOH(1.26 mmol, 1.00 eq) (2 mL) under N₂. The reaction mixture was stirredat 28° C. for 0.5 hr. LC-MS indicated the desired product was detected.The mixture was diluted with H₂O (10 mL) and MeOH (2 mL), washed withMTBE (10 mL×2). The resulting solution was diluted with H₂O and thenlyophilized to give a residue, which was purified by prep-HPLC(CH₃CN/H₂O/TFA) to give the product, but LC-MS indicated it was not pureenough. So it was further purified by prep-HPLC (CH₃CN/H₂O/TFA) to givea solution, which was lyophilized to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetralin-2-carboxamidetrifluoroacetate (52 mg) as a yellow solid. MS m/z=422.2 (MH⁺).

Example 27. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-methyl-cyclohexanecarboxamide(33)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-[(1-methylcyclohexanecarbonyl)-amino]-6-oxo-hexyl]carbamate(33a)

To a mixture of 1-methylcyclohexanecarboxylic acid (118 mg, 829.58 μmol,1.20 eq) and DIPEA (268 mg, 2.07 mmol, 3.00 eq) in THF (5 mL) was addedHATU (315 mg, 829.58 μmol, 1.20 eq) at 0° C., the reaction mixture wasstirred at 0° C. for 15 min. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added at 0° C., thereaction was stirred at 20° C. for another 3 h. LC-MS indicated thestarting material was consumed completely. The reaction was quenched byH₂O (10 mL) and extracted with EA (20 mL×2). The organic layers werecombined, washed with saturated brine (20 mL×2) and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-[(1-methylcyclohexanecarbonyl)amino]-6-oxo-hexyl]carbamate(704 mg, crude) as a yellow oil, which was used directly without furtherpurification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-methyl-cyclohexanecarboxamide(33)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-[(1-methylcyclohexanecarbonyl)amino]-6-oxo-hexyl]carbamate (704 mg, 1.26 mmol, 1.00 eq) in AcOH (3 mL)was added HBr/AcOH (1 mL, 33% purity). The reaction mixture was stirredat 20° C. for 3 h. LC-MS indicated the starting material was consumedcompletely. The reaction was quenched by H₂O (10 mL) and washed withMTBE (20 mL). The organic phase was extracted with H₂O (20 mL×2) and theaqueous phases were combined and lyophilized to give a crude, which waspurified by prep-HPLC (CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-methyl-cyclohexanecarboxamidetrifluoroacetate (50.68 mg, 101.04 μmol, 8.0% yield, 73.1% ee) as ayellow solid. MS m/z=388 (MH⁺). ¹H NMR (CD₃OD, 400 MHz) d 8.19 (d,J=7.6, 1H), 8.12 (d, J=7.6, 1H), 7.66-7.59 (m, 2H), 5.58 (dd, J=10.0,J=4.0, 1H), 3.05-2.94 (m, 2H), 2.23-2.14 (m, 1H), 2.08-2.00 (m, 2H),1.93-1.71 (m, 3H), 1.62-1.23 (m, 10H), 1.14 (s, 3H).

Example 28. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydrofuran-2-carboxamide(34/35)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydrofuran-2-carbonylamino)hexyl]carbamate(2)

To a mixture of tetrahydrofuran-2-carboxylic acid (96 mg, 829.58 μmol,1.20 eq) and DIPEA (268 mg, 2.07 mmol, 3.00 eq) in THF (5 mL) was addedHATU (315 mg, 829.58 μmol, 1.20 eq) at 0° C., the reaction mixture wasstirred at 0° C. for 15 min. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added at 0° C., themixture was stirred at 20° C. for another 3 h. LC-MS indicated thestarting material was consumed completely. The reaction was quenched byH₂O (10 mL) and extracted with EA (20 mL×2), the organic layers werecombined, washed with saturated brine (20 mL×2) and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydrofuran-2-carbonylamino)hexyl]carbamate(517 mg, crude) as a yellow oil, which was used without furtherpurification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydrofuran-2-carboxamide(34/35)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(tetrahydrofuran-2-carbonylamino)hexyl]carbamate(517 mg, 1.04 mmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (1 mL,33% purity). The reaction mixture was stirred at 20° C. for 1.5 h. LC-MSindicated the starting material was consumed completely. The reactionwas quenched by H₂O (10 mL) and washed with MTBE (20 mL). The organicphase was extracted with H₂O (20 mL×2), and the aqueous phases werecombined and lyophilized to give a crude, which was purified byprep-HPLC (CH₃CN/H₂O/TFA) to give two separated diastereomers ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]tetrahydrofuran-2-carboxamide trifluoroacetate as yellow solids.Compound 34 (33.78 mg, 71.04 μmol, 6.8% yield) MS m/z=362 (MH⁺). ¹H NMR(CD₃OD, 400 MHz) d 8.20 (d, J=7.6, 1H), 8.13 (d, J=7.6, 1H), 7.67-7.60(m, 2H), 5.65 (dd, J=9.2, J=4.0, 1H), 4.37 (dd, J=8.2, J=4.2, 1H),4.06-4.04 (m, 1H), 3.93-3.90 (m, 1H), 3.00-2.95 (m, 2H), 2.28-2.15 (m,2H), 1.97-1.81 (m, 4H), 1.77-1.65 (m, 2H), 1.59-1.51 (m, 2H). Compound35 (41.00 mg, 86.23 μmol, 8.3% yield) MS m/z=362 (MH⁺). ¹H NMR (CD₃OD,400 MHz) d 8.21 (dd, J=7.6, 1H), 8.13 (dd, J=7.6, 1H), 7.68-7.60 (m,2H), 5.71-5.64 (m, 1H), 4.39 (dd, J=8.2, J=5.4, 1H), 4.03 (q, J=7.0,1H), 3.89 (q, J=7.4, 1H), 3.01-2.95 (m, 2H), 2.30-2.15 (m, 2H),2.03-1.82 (m, 4H), 1.77-1.65 (m, 2H), 1.61-1.55 (m, 2H).

Example 29. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]thiazole-5-carboxamide(38)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(thiazole-5-carbonylamino)hexyl]carbamate(38a)

To a mixture of thiazole-5-carboxylic acid (107 mg, 829.58 μmol, 1.20eq) in THF (5 mL) were added HATU (315 mg, 829.58 μmol, 1.20 eq) andDIPEA (268 mg, 2.07 mmol, 3.00 eq) at 0° C., the reaction was stirred at0° C. for 15 min. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added at 0° C., thereaction mixture was stirred at 20° C. for another 3 h. LC-MS indicatedthe starting material was consumed completely. The reaction was quenchedby H₂O (10 mL), extracted with EA (20 mL×2). The organic layers werecombined, washed with saturated brine (20 mL×2) and evaporated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(thiazole-5-carbonylamino)hexyl]carbamate(497 mg, crude) as a red solid.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]thiazole-5-carboxamide(38)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(thiazole-5-carbonylamino)hexyl]carbamate(497 mg, 977.17 μmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (1 mL,33% purity) at 0° C. The mixture was stirred at 20° C. for 1 h. LC-MSindicated the reaction completed. H₂O (10 mL) was added, the solutionwas wash with MTBE (20 mL). The organic phase was extracted with H₂O (20mL×2) and the aqueous phases were combined and lyophilized to give aresidue, which was purified by prep-HPLC (CH₃CN/H₂O/TFA), to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]thiazole-5-carboxamidetrifluoroacetate (139.46 mg, 285.49 μmol, 29.2% yield, 82.2% ee) as ayellow solid. MS m/z=375.1 (MH⁺).

Example 30. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]thiazole-2-carboxamide(39)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(thiazole-2-carbonylamino)hexyl]carbamate(39a)

To a solution of thiazole-2-carboxylic acid (110 mg, 851.79 μmol, 1.23eq) in THF (6 mL) were added HATU (390 mg, 1.03 mmol, 1.48 eq) and DIPEA(270 mg, 2.09 mmol, 3.02 eq) under 0° C. After being stirred for 0.5 hunder 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq, HCl) was added and theresulting mixture was stirred at 28° C. for 14 h. It was diluted withH₂O (10 mL) and extracted with EA (15 mL×2). The organic layers werecombined, dried over Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(thiazole-2-carbonylamino)hexyl]carbamate(510 mg, crude) as a yellow solid. It was used directly for the nextstep without further purification.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]thiazole-2-carboxamide(39)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-(thiazole-2-carbonylamino)hexyl]carbamate(1.00 mmol, 1.00 eq) (0.51 g, crude) in AcOH (5 mL) was added HBr/AcOH(1.00 mmol, 1.00 eq) (2 mL, 33% purity) under N₂. The reaction mixturewas stirred at 28° C. for 1 hr. LC-MS indicated the desired product wasdetected. The mixture was diluted with H₂O (10 mL) and MeOH (2 mL),washed with MTBE (10 mL×2). The resulting solution was diluted with H₂O(80 mL) and then lyophilized to give a residue, which was purified byprep-HPLC (CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]thiazole-2-carboxamidetrifluoroacetate (20 mg) as a yellow solid. MS m/z=375.0 (MH⁺).

Example 31. Preparation ofN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-6-oxo-1H-pyridine-2-carboxamide(40)

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[(6-oxo-1H-pyridine-2-carbonyl)amino]hexyl]carbamate(40a)

A solution of 6-oxo-1H-pyridine-2-carboxylic acid (115 mg, 829.59 μmol,1.20 eq), DIPEA (268 mg, 2.07 mmol, 3.00 eq) and HATU (315 mg, 829.58μmol, 1.20 eq) in THF (3 mL) was stirred at 0° C. for 15 min. Thenbenzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo -hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added at 0° C., theresulting mixture was stirred at 25° C. for another 3 h. LC-MS indicatedthe reaction completed. H₂O (10 mL) was added and extracted with EA (20mL×2). The combined organic phase was washed with saturated brine (20mL) and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[(6-oxo-1H-pyridine-2-carbonyl)amino]hexyl]carbamate(612 mg, crude) as a yellow solid.

N-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-6-oxo-1H-pyridine-2-carboxamide(40)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-6-oxo-5-[(6-oxo-1H-pyridine-2-carbonyl)amino]hexyl]carbamate(612 mg, 1.18 mmol, 1.00 eq) in AcOH (3 mL) was added HBr/AcOH (1 mL,33% purity) at 0° C. The reaction mixture was stirred at 20° C. for 1 h.LC-MS indicated the desired compound was detected. The reaction wasquenched by H₂O (10 mL), washed with MTBE (20 mL). The organic phase wasextracted with H₂O (20 mL×2) and the aqueous phases were combined andlyophilized to give crude product, which was purified by prep-HPLC(CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-6-oxo-1H-pyridine-2-carboxamidetrifluoroacetate (51.45 mg, 103.22 μmol, 80.4% yield, 74.1% ee) as ayellow solid. MS m/z=385.1 (MH⁺).

Example 32. Preparation of(S)—N-(6-amino-1-(benzo[d]thiazol-2-yl)-1-oxohexan-2-yl)-1-methoxycyclohexanecarboxamide(41)

1-methoxycyclohexanecarboxylic Acid (41b)

To a solution of cyclohexanone (2.00 g, 20.38 mmol, 1.00 eq) in CHCl3(2.43 g, 20.38 mmol, 1.00 eq) (10 mL) was added a solution of KOH (10.00g, 178.22 mmol, 8.74 eq) in MeOH (10 mL) at 0-5° C. The resultingmixture was stirred at 23° C. for 3 hr. The suspension was filtered andthe filtrate was concentrated to give a crude, which was diluted withH₂O (20 mL) and extracted with EA (20 mL×2). Then the aqueous layer wasadjusted to pH=5-6 with HCl (1 M). The resulting solution was extractedwith DCM (40 mL×2). The organic layers were combined, dried over Na₂SO₄,filtered and concentrated to give 1-methoxycyclohexanecarboxylic acid(1.21 g, 7.65 mmol, 37.53% yield) as a yellow oil.

Benzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-[(1-methoxycyclohexanecarbonyl)amino]-6-oxo-hexyl]carbamate(41a)

To a solution of 1-methoxycyclohexanecarboxylic acid (164 mg, 1.04 mmol,1.50 eq) in THF (6 mL) were added HATU (390 mg, 1.03 mmol, 1.48 eq) andDIPEA (300 mg, 2.32 mmol, 3.36 eq) under 0° C. After being stirred for0.5 h under 0° C.,benzyl-N-[(5S)-5-amino-6-(1,3-benzothiazol-2-yl)-6-oxo-hexyl]carbamatehydrochloride (300 mg, 691.32 μmol, 1.00 eq) was added and the resultingmixture was stirred at 20° C. for 14 h. It was diluted with H₂O (10 mL)and extracted with EA (15 mL×2). The organic layers were combined, driedover Na₂SO₄, filtered and concentrated to givebenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-[(1-methoxycyclo-hexanecarbonyl)amino]-6-oxo-hexyl]carbamate(830 mg, crude) as a yellow solid. It was used directly for the nextstep without further purification.

(S)—N-(6-amino-1-(benzo[d]thiazol-2-yl)-1-oxohexan-2-yl)-1-methoxy-cyclohexanecarboxamide(41)

To a solution ofbenzyl-N-[(5S)-6-(1,3-benzothiazol-2-yl)-5-[(1-methoxycyclohexanecarbonyl)amino]-6-oxo-hexyl]carbamate (1.00 eq) (0.83 g, crude) in AcOH (5 mL)was added HBr/AcOH (1.00 eq) (1.5 mL, 33% purity) under N₂. The reactionmixture was stirred at 18° C. for 1 hr. The mixture was diluted with H₂O(100 mL) and washed with MBTE (40 mL). The aqueous layer was lyophilizedto give a residue, which was purified by prep-HPLC (CH₃CN/H₂O/TFA) togive a solution. The solution was lyophilized to giveN-[(1S)-5-amino-1-(1,3-benzothiazole-2-carbonyl)pentyl]-1-methoxy-cyclohexanecarboxamidetrifluoroacetate (55 mg, 106.27 μmol) as a light yellow solid. MSm/z=404.2 (MH⁺).

Example 33. Preparation ofN-[(1S)-5-amino-1-(thiazole-2-carbonyl)pentyl]cyclopentanecarboxamide(42)

Benzyl-N-[(5S)-5-amino-6-oxo-6-thiazol-2-yl-hexyl]carbamate (42b)

To a solution oftert-butyl-N-[(1S)-5-(benzyloxycarbonylamino)-1-(thiazole-2-carbonyl)pentyl]carbamate(300 mg, 670.32 μmol, 1.00 eq) in EA (10 mL) was added HCl/EA (4 M, 2mL, 11.93 eq). The reaction mixture was stirred at 10° C. for 14 hr.LC-MS indicated the starting material was still remained and the desiredproduct was detected. HCl/EA (4 M, 2.00 mL, 11.93 eq) was added. Thenthe reaction mixture was stirred at 10° C. for another 4 hr. TLC(PE:EA=3:1) indicated the starting material was consumed completely. Itwas concentrated to givebenzyl-N-[(5S)-5-amino-6-oxo-6-thiazol-2-yl-hexyl]carbamatehydrochloride (310 mg, crude, HCl) as a brown solid. It was useddirectly for next step without further purification.

Benzyl-N-[(5S)-5-amino-6-oxo-6-thiazol-2-yl-hexyl]carbamate (42a)

To a solution ofbenzyl-N-[(5S)-5-amino-6-oxo-6-thiazol-2-yl-hexyl]carbamatehydrochloride (300 mg, 781.47 μmol, 1.00 eq, HCl) in DCM (10 mL) wasadded TEA (240 mg, 2.37 mmol, 3.04 eq) and cyclopentanecarbonyl chloride(130 mg, 980.47 μmol, 1.25 eq). The reaction mixture was stirred at 10°C. for 3 hr. LC-MS indicated the starting material was consumed and themajor was desired product. The solution was washed with H₂O (15 mL×2).The aqueous layer was extracted with DCM (20 mL×2). The organic layerswere combined, washed with sat. brine (100 mL), dried over Na₂SO₄,filtered and concentrated to give a crude. The crude was purified bycolumn chromatography (PE:EA=4: 1 to 2: 1) to givebenzyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-6-thiazol-2-yl-hexyl]carbamate(230 mg, 482.23 μmol, 61.7% yield, 93% purity) as a yellow solid.

N-[(1S)-5-amino-1-(thiazole-2-carbonyl)pentyl]cyclopentanecarboxamide(42)

To a solution ofbenzyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-6-thiazol-2-yl-hexyl]carbamate (210 mg, 473.44 μmol, 1.00 eq) in AcOH (5 mL) was addedHBr/AcOH (2 mL, 33% purity) under N₂. The reaction mixture was stirredat 15° C. for 1 hr. LC-MS indicated the starting material was remainedand the desired product was detected. The mixture was stirred at 15° C.for another 1 hr. LC-MS indicated the starting material was stillremained and the desired product was detected. The mixture was deionizedwater (20 mL) and MeOH (2 mL), washed with MTBE (20 mL×2). The resultingsolution was diluted with deionized water (80 mL) and then lyophilizedto give a residue, which was adjusted to pH=7-8 with sat. aq. NaHCO₃.The resulting solution was diluted with deionized water (80 mL) and thenlyophilized again to give a residue, which was purified by prep-HPLC(CH₃CN/H₂O/TFA) to giveN-[(1S)-5-amino-1-(thiazole-2-carbonyl)pentyl]cyclopentanecarboxamidetrifluoroacetate (53 mg) as a yellow solid. MS m/z=304.2 (MH⁺).

Example 34. Preparation ofN-[(1S)-5-amino-1-[2-(2,3,5,6-tetrafluorophenoxy)acetyl]-pentyl]cyclopentanecarboxamide(43)

(2S)-6-(tert-butoxycarbonylamino)-2-(cyclopentanecarbonylamino)hexanoicacid (43d)

To a solution of (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoic acid(5.00 g, 20.30 mmol, 1.00 eq) in H₂O (40 mL) were added NaOH (820 mg,20.50 mmol, 1.01 eq) and Na₂CO₃ (2.58 g, 24.36 mmol, 1.20 eq). Afterbeing stirred for 10 min, it was cooled to 0° C. andcyclopentanecarbonyl chloride (2.96 g, 22.33 mmol, 1.10 eq) in EA (20mL) was added under 0° C. The reaction mixture was stirred at 10° C. for14 h. EA was removed and then solution was cooled to 0° C., the pH wasadjusted to 6-7 with solid KHSO₄. The suspension was filtered and thefilter cake was washed with PE (60 mL) and dried in vacuo to give(2S)-6-(tert-butoxycarbonylamino)-2-(cyclopentanecarbonylamino)hexanoicacid (4.50 g, crude) as a white solid.

Tert-butyl-N-[(5S)-5-(cyclopentanecarbonylamino)-7-diazo-6-oxo-heptyl]carbamate(43c)

To a solution of(2S)-6-(tert-butoxycarbonylamino)-2-(cyclopentane-carbonylamino)hexanoicacid (2.00 g, 5.84 mmol, 1.00 eq) in THF (20 mL) was added isobutylchloroformate (798 mg, 5.84 mmol, 1.00 eq) and NMM (591 mg, 5.84 mmol,1.00 eq). The mixture was stirred at −20° C. for 2 h under N₂. Thendiazomethane (491 mg, 11.68 mmol, 2.00 eq) was added. The mixture wasstirred at 0° C. for 10 h. The mixture was diluted with H₂O (20 mL),extracted with EA (20 mL×3). The organic layers were combined, driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue, which was purified by silica gel chromatography (PE/EA=1/1) togivetert-butyl-N-[(5S)-5-(cyclopentanecarbonylamino)-7-diazo-6-oxo-heptyl]carbamate(2.00 g, 5.46 mmol, 93.4% yield) as a yellow solid.

Tert-butyl-N-[(5S)-7-bromo-5-(cyclopentanecarbonylamino)-6-oxoheptyl]carbamate(43b)

To a solution oftert-butyl-N-[(5S)-5-(cyclopentanecarbonylamino)-7-diazo-6-oxo-heptyl]carbamate (1.00 g, 2.73 mmol, 1.00 eq) in EA (20 mL) was added HBr/AcOH(Purity: 33%, 1 mL). The mixture was stirred at −20° C. for 10 min underN₂ and then basified with sat. NaHCO₃ till pH=8, extracted with EA (20mL×3). The organic layers were combined, dried over Na₂SO₄, filtered andconcentrated to givetert-butyl-N-[(5S)-7-bromo-5-(cyclopentanecarbonylamino)-6-oxoheptyl]carbamate(1.10 g, 2.62 mmol, 96.08% yield) as a yellow solid.

Tert-butyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-7-(2,3,5,6-tetrafluorophenoxy)heptyl]carbamate(43a)

To a solution oftert-butyl-N-[(5S)-7-bromo-5-(cyclopentanecarbonylamino)-6-oxo-heptyl]carbamate (1.10 g, 2.62 mmol, 1.00 eq) in DMF (20 mL) was added2,3,5,6-tetrafluorophenol (523 mg, 3.15 mmol, 1.20 eq) and KF (457 mg,7.87 mmol, 3.00 eq). The reaction mixture was stirred at 20° C. for 3 h.The mixture was diluted with H₂O (50 mL) and extracted with EA (40mL×3). The organic layers were combined, washed with brine (50 mL×5),dried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography (PE/EA=4/1) to givetert-butyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-7-(2,3,5,6-tetrafluorophenoxy)heptyl]carbamate(1.10 g, 2.18 mmol, 83.2% yield) as a white solid.

N-[(1S)-5-amino-1-[2-(2,3,5,6-tetrafluorophenoxy)acetyl]pentyl]cyclopentanecarboxamide(43)

To a solution oftert-butyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-7-(2,3,5,6-tetrafluorophenoxy)heptyl]carbamate(500 mg, 991.06 μmol, 1.00 eq) in EA (10 mL) was added HCl/EA (4 M, 5mL, 20.18 eq). The mixture was stirred at 20° C. for 1 h. The mixturewas diluted with H₂O (20 mL), the aqueous layer was separated andlyophilized to giveN-[(1S)-5-amino-1-[2-(2,3,5,6-tetrafluorophenoxy)acetyl]pentyl]cyclopentanecarboxamidehydrochloride (280 mg, 635.12 μmol, 64.1% yield) as a white solid. MSm/z=405 (MH⁺). ¹H NMR ((CD₃)₂SO, 400 MHz) d 8.38 (d, J=7.2, 1H), 8.05(bs, 3H), 7.58 (ddd, J=4.5, J=8.3, J=15.5, 1H), 5.25 (d, J=17.6, 1H),5.18 (d, J=17.6, 1H), 4.29 (ddd, J=2.4, J=4.6, J=10.6, 1H), 2.77-2.64(m, 3H), 1.78-1.24 (m, 14H).

Example 35. Preparation ofN-[(1S)-5-amino-1-(pyridine-2-carbonyl)pentyl]cyclopentane-carboxamide(44)

Benzyl-N-[(5S)-5-amino-6-oxo-6-(2-pyridyl)hexyl]carbamate (44b)

To a solution oftert-butyl-N-[(1S)-5-(benzyloxycarbonylamino)-1-(pyridine-2-carbonyl)pentyl]carbamate(480 mg, 1.09 mmol, 1.00 eq) in EA (10 mL) was added HCl/EA (4 M, 4 mL,14.68 eq). The reaction mixture was stirred at 10° C. for 14 hr. LC-MSindicated the starting material was consumed completely and the desiredproduct was detected. It was concentrated to givebenzyl-N-[(5S)-5-amino-6-oxo-6-(2-pyridyl)hexyl]carbamate hydrochloride(400 mg, crude) as a yellow solid. It was used directly for next stepwithout further purification.

Benzyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-6-(2-pyridyl)-hexyl]carbamate(44a)

To a solution ofbenzyl-N-[(5S)-5-amino-6-oxo-6-(2-pyridyl)hexyl]carbamate hydrochloride(400 mg, 1.06 mmol, 1.00 eq) in DCM (10 mL) was added TEA (320 mg, 3.16mmol, 2.98 eq) and cyclopentanecarbonyl chloride (160 mg, 1.21 mmol,1.14 eq). The reaction mixture was stirred at 10° C. for 3 hr. LC-MSindicated the starting material was consumed and the major was thedesired product. The solution was washed with H₂O (15 mL×2). The aqueouslayer was extracted with DCM (20 mL×2). The organic layers werecombined, washed with sat. brine (100 mL), dried over Na₂SO₄, filteredand concentrated to give a crude, which was purified by columnchromatography (PE: EA=4: 1 to 2: 1) to givebenzyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-6-(2-pyridyl)hexyl]carbamate(250 mg, 571.39 μmol, 53.9% yield) as a light yellow solid.

N-[(1S)-5-amino-1-(pyridine-2-carbonyl)pentyl]cyclopentanecarboxamide(44)

To a solution ofbenzyl-N-[(5S)-5-(cyclopentanecarbonylamino)-6-oxo-6-(2-pyridyl)hexyl]carbamate (230 mg, 525.68 μmol, 1.00 eq) in AcOH (5 mL) was addedHBr/AcOH (2 mL, 33% purity) under N₂. The reaction mixture was stirredat 15° C. for 1 hr. LC-MS indicated the starting material was stillremained and the desired product was detected. The reaction mixture wasstirred at 15° C. for another 1 hr. LC-MS indicated the startingmaterial was still remained and the desired product was detected. Themixture was diluted with deionized water (20 mL) and MeOH (2 mL), washedwith MTBE (20 mL×2). The resulting solution was diluted with deionizedwater (80 mL) and then lyophilized to give a residue, which was adjustedto pH=7-8 with sat. aq. NaHCO₃. The resulting solution was diluted withdeionized water (80 mL) and then lyophilized again to give a residue. Apart of crude was purified by prep-HPLC (CH₃CN/H₂O/NH₃.H₂O) to give thedesired product but it contained cyclized product. Other part of crudewas purified by prep-HPLC (CH₃CN/H₂O/TFA) toN-[(1S)-5-amino-1-(pyridine-2-carbonyl)pentyl]cyclopentanecarboxamidetrifluoroacetate (56 mg) as a yellow solid. MS m/z=304.2 (MH⁺).

Example 36. Preparation ofN-[(1S)-5-amino-1-[2-(2,3,5,6-tetrafluorophenoxy)acetyl]-pentyl]-3-azido-benzamide(45)

(2S)-2-[(3-azidobenzoyl)amino]-6-(tertbutoxycarbonylamino)hexanoic acid(45d)

To a solution of (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoic acid(10.00 g, 40.60 mmol, 1.00 eq) in EA/H₂O (1/1, 200 mL) were added NaOH(1.62 g, 40.60 mmol, 1.00 eq), Na₂CO₃ (4.30 g, 40.60 mmol, 1.00 eq) and3-azidobenzoyl chloride (7.37 g, 40.60 mmol, 1.00 eq). The mixture wasstirred at 20° C. for 4 h. The mixture was acidified with KHSO₄ tillpH=4, concentrated under reduced pressure. The residue was purified bysilica gel chromatography eluted with DCM/MeOH=10/1 to give(2S)-2-[(3-azidobenzoyl)amino]-6-(tertbutoxycarbonylamino) hexanoic acid(10.00 g, 25.55 mmol, 62.9% yield) as a yellow solid.

Tert-butyl-N-[(5S)-5-[(3-azidobenzoyl)amino]-7-diazo-6-oxo-heptyl]carbamate(45c)

To a solution of(2S)-2-[(3-azidobenzoyl)amino]-6-(tert-butoxycarbonylamino)hexanoic acid(3.50 g, 8.94 mmol, 1.00 eq) in THF (50 mL) was added NMM (904 mg, 8.94mmol, 1.00 eq) and isobutyl chloroformate (1.22 g, 8.94 mmol, 1.00 eq).The mixture was stirred at −20° C. for 1 h under N₂. Then diazomethane(376 mg, 8.94 mmol, 1.00 eq) was added. The mixture was stirred at −20°C. for 4 h under N₂. The mixture was diluted with H₂O (50 mL), extractedwith EA (50 mL×3). The organic layers were combined, dried over Na₂SO₄,filtered and concentrated under reduced pressure. The residue waspurified by silica gel chromatography eluted with PE/EA=1/1 to givetert-butyl-N-[(5S)-5-[(3-azidobenzoyl)amino]-7-diazo-6-oxo-heptyl]carbamate(2.90 g, 6.98 mmol, 78.1% yield) as yellow solid.

Tert-butyl-N-[(5S)-5-[(3-azidobenzoyl)amino]-7-bromo-6-oxo-heptyl]-carbamate(45b)

To a solution of(3-azidobenzoyl)-[(1S)-5-(tert-butoxycarbonylamino)-1-(2-diazoacetyl)pentyl]ammonium(2.90 g, 6.96 mmol, 1.00 eq) in EA (50 mL) was added HBr/AcOH (2.56 g,10.44 mmol, 1.50 eq, Purity: 33%). The mixture was stirred at −20° C.for 10 min. The mixture was diluted with H₂O (50 mL), extracted with EA(50 mL). The organic layer was washed with brine (50 mL×3), dried overNa₂SO₄, filtered and concentrated under reduced pressure to givetert-butyl-N-[(5S)-5-[(3-azidobenzoyl)amino]-7-bromo-6-oxo-heptyl]carbamate(2.90 g, crude, Purity: 62.2%) as a yellow solid.

Tert-butyl-N-[(5S)-5-[(3-azidobenzoyl)amino]-6-oxo-7-(2,3,5,6-tetrafluoro-phenoxy)heptyl]carbamate(45a)

To a solution oftert-butyl-N-[(5S)-5-[(3-azidobenzoyl)-amino]-7-bromo-6-oxo-heptyl]carbamate(2.90 g, 6.19 mmol, 1.00 eq) in DMF (20 mL) were added2,3,5,6-tetrafluorophenol (1.54 g, 9.29 mmol, 1.50 eq) and KF (1.80 g,30.95 mmol, 5.00 eq). The mixture was stirred at 20° C. for 12 h. Themixture was diluted with H₂O (100 mL), extracted with EA (100 mL). Theorganic layer was washed with brine (100 mL×5), dried over Na₂SO₄,filtered and concentrated under reduced pressure. The residue waspurified by silica gel chromatography eluted with PE:EA=4:1 to givetert-butyl-N-[(5S)-5-[(3-azidobenzoyl)amino]-6-oxo-7-(2,3,5,6-tetrafluorophenoxy)heptyl]carbamate(2.80 g, 5.06 mmol, 81.7% yield) as a white solid.

N-[(1S)-5-amino-1-[2-(2,3,5,6-tetrafluorophenoxy)acetyl]pentyl]-3-azido-benzamide(45)

A solution oftert-butyl-N-[(5S)-5-[(3-azidobenzoyl)amino]-6-oxo-7-(2,3,5,6-tetrafluorophenoxy)heptyl]carbamate(2.20 g, 3.97 mmol, 1.00 eq) in HO/EA (20 mL, 4 M) was stirred at 20° C.for 30 min. The mixture was diluted with H₂O (20 mL), the aqueous layerwas lyophilized to giveN-[(1S)-5-amino-1-[2-(2,3,5,6-tetrafluorophenoxy)acetyl]pentyl]-3-azido-benzamideas a white solid. MS m/z=454.1 (MH⁺).

Example 37. Inhibition of Lysine Gingipain by Compounds of the Invention

The capacities of compounds of the present invention to inhibit theactivity of lysine gingipain were measured in a fluorogenic assaysimilar to those described in Barret Biochemical Journal. 1980, 187(3),909. The specific assay conditions were as follows. Buffer: pH=7.5, 100mM Tris-HCl, 75 mM NaCl, 2.5 mM CaCl2, 10 mM cysteine, 1% DMSO after alladditions. Protein: 0.1 nM Kgp, isolated from culture of Porphyromonasgingivalis, as described in Pike et al. J. Biol. Chem. 1994, 269(1),406, and Potempa and Nguyen. Current Protocols in Protein Scienc. 2007,21.20.1-21.20.27. Fluorogenic substrate: 10 uM Z-His-Glu-Lys-MCA.Time=90 minutes. Temperature=37° C. Each compound: 10 concentrations,starting at either 100 uM or 100 nM, with lower concentrations generatedby serial 3-fold dilutions. By testing a range of concentrations foreach compound, the concentration required to inhibit the activity oflysine gingipain by 50% (the “IC₅₀”) was determined. Under the describedassay conditions, signal-to-noise was excellent, and Z factor wasgreater than 0.6.

The inhibitory of activity of compounds described herein was testedagainst Kgp, RgpB, and trypsin. Kgp IC₅₀ values for various compoundsare set forth in Table 2. Compound 43 exhibited an RgpB IC₅₀ value above475 nM, and the remaining compounds in Table 2 exhibited RgpB IC₅₀values above 2.5 μM. All of the compounds in Table 2 exhibited trypsinIC₅₀ values above 1 μM.

TABLE 2 Lysine gingipain inhibitory activity of compounds of theinvention. Compound No. Kgp IC₅₀ average 1 +++ 1 * 2 ** 2 *** 3 ** 4 ***7 * 8 ** 9 *** 10 ** 11 *** 12 +++ 13 * 14 *** 15 *** 18 ** 19 ** 22 **23 ++ 24 *** 25 * 26 * 29 ** 30 * 31 ** 32 *** 33 *** 34 *** 35 *** 38** 39 +++ 40 * 41 ** 42 +++ 43 **** 44 + **** IC₅₀ ≤ 1 nM *** 1 nM <IC₅₀ ≤ 10 nM ** 10 nM < IC₅₀ ≤ 25 nM * 25 nM < IC₅₀ ≤ 50 nM +++ 50 nM <IC₅₀ ≤ 100 nM ++ 100 nM < IC₅₀ ≤ 250 nM ++ 250 nM < IC₅₀

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

1-45. (canceled)
 46. A pharmaceutical composition comprising a compoundaccording to Formula Ib:

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient, wherein Z is halogen-substitutedaryloxymethyl-carbonyl; A is selected from the group consisting of —CH₂—and —O—; B and D are independently selected from the group consisting ofhydrogen, halogen, halomethyl, and halomethoxy; R¹ is selected from thegroup consisting of hydrogen and an amine protecting group; R² ishydrogen; and R³ is selected from the group consisting of C₆₋₁₀ aryl,5-to-12 membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturatedheterocyclyl, and -L-R⁵, wherein L is selected from the group consistingof —O—, —NR—, C₁₋₄ alkylene, and 2- to 4-membered heteroalkylene,wherein R is selected from the group consisting of hydrogen and C₁₋₈alkyl, R⁵ is selected from the group consisting of C₆₋₁₀ aryl, 5-to-12membered heteroaryl, C₃₋₈ cycloalkyl, and 5-to-12 membered saturatedheterocyclyl, and wherein R³ is optionally substituted with one or moresubstituents selected from the group consisting of halo, —CN, —NO₂, —N₃,—OH, R^(a), R^(b), —OR^(a), —OR^(b), —(CH₂)_(k)C(O)R^(c),—NR^(d)(CH₂)_(u)C(O)R^(c), —O(CH₂)_(u)C(O)R^(c),—(CH₂)_(k)CONR^(d)R^(d), —(CH₂)_(k)NR^(d)C(O)R^(c),—NR^(d)(CH₂)_(u)CONR^(d)R^(d), —NR^(d)(CH₂)_(u)NR^(d)C(O)R^(c),—O(CH₂)_(u)CONR^(d)R^(d), —O(CH₂)_(u)NR^(d)C(O)R^(c),—(CH₂)_(k)S(O)₂NR^(d)R^(d), —(CH₂)_(k)NR^(d)S(O)₂R^(c),—(CH₂)_(k)S(O)₂R^(c), —(CH₂)_(k)S(O)R^(c), —(CH₂)_(k)SR^(d),—NR^(d)(CH₂)_(u)S(O)₂NR^(d)R^(d), —NR^(d)(CH₂)_(u)NR^(d)S(O)₂R^(c),—NR^(d)(CH₂)_(u)S(O)₂R^(c), —NR^(d)(CH₂)_(u)S(O)R^(c),—NR^(d)(CH₂)_(u)SR^(d), —O(CH₂)_(u)S(O)₂NR^(d)R^(d),—O(CH₂)_(u)NR^(d)S(O)₂R^(c), —O(CH₂)_(u)S(O)₂R^(c),—O(CH₂)_(u)S(O)R^(c), and —O(CH₂)_(u)SR^(c), wherein: each R^(a) isindependently selected from the group consisting of C₁₋₄ alkyl and C₁₋₄haloalkyl, each R^(b) is independently selected from the groupconsisting of C₃₋₆ cycloalkyl, C₃₋₆ halocycloalkyl, C₆₋₁₀ aryl, 5-to-12membered heteroaryl, and 5-to-12 membered saturated heterocyclyl, eachR^(c) is independently selected from the group consisting of —OH, C₁₋₈alkyl, C₁₋₈ haloalkyl, C₃₋₈ cycloalkyl, C₃₋₈ halocycloalkyl, C₆₋₁₀ aryl,(C₆₋₁₀ aryl)-(C₁₋₈ alkyl), 5-to-12 membered heteroaryl, and 5-to-12membered saturated heterocyclyl, each R^(d) is independently selectedfrom the group consisting of hydrogen and C₁₋₈ alkyl, each subscript kis independently selected from 0, 1, 2, 3, 4, 5, and 6, and eachsubscript u is independently selected from 1, 2, 3, 4, 5, and 6; and R⁴is selected from the group consisting of hydrogen, halogen, C₁₋₄ alkyl,C₁₋₄ alkoxy, C₁₋₄ haloalkyl, and C₁₋₄ haloalkoxy; provided that when Ais —CH₂—, and B and D are hydrogen, then R³ is other than(2-phenyl)ethyl or substituted (2-phenyl)ethyl.
 47. The pharmaceuticalcomposition of claim 46, wherein Z is halogen-substituted phenoxymethylcarbonyl.
 48. The pharmaceutical composition of claim 46, wherein R³ isselected from the group consisting of cyclohexyl, cyclopentyl,morpholino, phenyl, piperidinyl, pyridinyl, tetrahydrofuranyl,tetrahydropyranyl, 1,2,3,4-tetrahydronaphthyl, and thiazolyl, each ofwhich is optionally substituted with 1-3 members selected from the groupconsisting of methyl, methoxy, trifluoromethyl, acetyl, and —N₃.
 49. Thepharmaceutical composition of claim 46, wherein the compound is acompound, or pharmaceutically acceptable salt thereof, according toFormula Id:

wherein R³ is selected from the group consisting of C₆₋₁₀ aryl, 5-to-12membered heteroaryl, C₃₋₈ cycloalkyl, 5-to-12 membered saturatedheterocyclyl, and -L-R⁵, wherein L is C₁₋₄ alkylene.
 50. Thepharmaceutical composition of claim 49, wherein R³ is selected from thegroup consisting of cyclohexyl, cyclopentyl, morpholino, phenyl,piperidinyl, pyridinyl, tetrahydrofuranyl, tetrahydropyranyl,1,2,3,4-tetrahydronaphthyl, and thiazolyl, each of which is optionallysubstituted with 1-3 members selected from the group consisting ofmethyl, methoxy, trifluoromethyl, acetyl, and —N₃.
 51. Thepharmaceutical composition of claim 46, wherein the compound is selectedfrom the group consisting of:

and pharmaceutically acceptable salts thereof.
 52. The pharmaceuticalcomposition of claim 46, wherein the compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 53. The pharmaceuticalcomposition of claim 46, wherein the compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 54. The pharmaceuticalcomposition of claim 46, wherein the compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 55. The pharmaceuticalcomposition of claim 46, wherein R⁴ is selected from the groupconsisting of C₁₋₄ alkyl and C₁₋₄ haloalkyl.
 56. The pharmaceuticalcomposition of claim 55, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 57. A pharmaceuticalcomposition comprising a compound according to the formula:

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient, wherein Z is selected from the group consisting ofbenzothiazol-2-yl-carbonyl, pyridin-2-yl-carbonyl, andthiazol-2-yl-carbonyl; and R³ is selected from the group consisting ofcyclohexyl; 1-methylcyclohexyl; 1-methoxycyclohexyl; cyclopentyl;morpholin-2-yl; 4-acetylmorpholin-2-yl; phenyl; 2-trifluoromethylphenyl;3-azidophenyl; piperidine-3-yl; 1-acetyl-piperidine-3-yl; pyridin-2-yl;pyridin-3-yl; pyridin-4-yl; 6-oxo-1,6-dihydropyridin-2-yl;tetrahydrofuran-2-yl; tetrahydro-2H-pyran-2-yl;tetrahydro-2H-pyran-3-yl; tetrahydro-2H-pyran-4-yl;1,2,3,4-tetrahydronaphth-1-yl; 1,2,3,4-tetrahydronaphth-2-yl;thiazol-5-yl; and thiazol-2-yl.
 58. The pharmaceutical composition ofclaim 57, wherein the compound is a compound, or pharmaceuticallyacceptable salt thereof, according to the formula:


59. The pharmaceutical composition of claim 58, wherein R³ is selectedfrom the group consisting of cyclohexyl; 1-methylcyclohexyl;1-methoxycyclohexyl; cyclopentyl; morpholin-2-yl;4-acetylmorpholin-2-yl; phenyl; 2-trifluoromethylphenyl; 3-azidophenyl;piperidine-3-yl; 1-acetyl-piperidine-3-yl; pyridin-2-yl; pyridin-3-yl;pyridin-4-yl; 6-oxo-1,6-dihydropyridin-2-yl; tetrahydrofuran-2-yl;tetrahydro-2H-pyran-2-yl; tetrahydro-2H-pyran-3-yl;tetrahydro-2H-pyran-4-yl; 1,2,3,4-tetrahydronaphth-1-yl;1,2,3,4-tetrahydronaphth-2-yl; thiazol-5-yl; and thiazol-2-yl.
 60. Thepharmaceutical composition of claim 57, wherein the compound is selectedfrom the group consisting of:

and pharmaceutically acceptable salts thereof.
 61. The pharmaceuticalcomposition of claim 57, wherein the compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 62. The pharmaceuticalcomposition of claim 57, wherein Z is selected from the group consistingof pyridin-2-yl-carbonyl and thiazol-2-yl-carbonyl.
 63. Thepharmaceutical composition of claim 62, wherein the compound is selectedfrom the group consisting of:

and pharmaceutically acceptable salts thereof.
 64. The pharmaceuticalcomposition of claim 62, wherein the compound is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.